EP1613780A2 - New process for improving the manufacture of clavams e.g. clavulanic acid - Google Patents

New process for improving the manufacture of clavams e.g. clavulanic acid

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Publication number
EP1613780A2
EP1613780A2 EP04727009A EP04727009A EP1613780A2 EP 1613780 A2 EP1613780 A2 EP 1613780A2 EP 04727009 A EP04727009 A EP 04727009A EP 04727009 A EP04727009 A EP 04727009A EP 1613780 A2 EP1613780 A2 EP 1613780A2
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EP
European Patent Office
Prior art keywords
seq
clavulanic acid
orβ
open reading
polynucleotide
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP04727009A
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German (de)
French (fr)
Inventor
Barry Barton
Susan The Govern. of the Univ. of Alberta JENSEN
Alison Michelle Griffin
Annie The Governors of the Univ. of Alberta WONG
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University of Alberta
Glaxo Group Ltd
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University of Alberta
Glaxo Group Ltd
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Publication of EP1613780A2 publication Critical patent/EP1613780A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the present invention relates to new processes for improving the manufacture of clavams e.g. clavulanic acid.
  • the present invention also provides novel DNA sequences and new microorganisms capable of producing increased amounts of clavulanic acid.
  • Microorganisms in particular Streptomyces sp. produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins, polyketides, cephamycins, tunicamycin, holomycin and penicillins.
  • antibiotics including clavulanic acid and other clavams, cephalosporins, polyketides, cephamycins, tunicamycin, holomycin and penicillins.
  • Streptomyces clavuligerus produces two major groups of antibiotics; one being the cephamycins, cephalosporins and penicillins (Demain, A.L. (1990) supra) and the other comprising clavams.
  • Clavams can be arbitrarily divided into two groups, 5S and 5R clavams, dependent on their ring stereochemistry.
  • Examples of 5S clavams are clavam-2-carboxylate (C-2-C), 2-hydroxymethyl clavam (2HMC) and alanylclavam (Brown et al. (1979) J. Chem.
  • the Soy media contains very small amounts one or more of the metabolites produced by the orfs 2-6 allowing strains disrupted in these genes to make small amounts of clavulanic acid.
  • Marsh et al. (1992) supra has reported that S. clavuligerus contains two copies of the cas gene (casl and cas2).
  • casl is not associated with the clavulanic acid gene cluster and has a high homology to casl. Disruption of casl decreases clavulanic acid production by 35% when cultures are grown on Soy medium and eliminates production entirely when cultures are grown on starch asparagine (SA) medium (Paradkar and Jensen 1995 J.Bact 177: 1307- 1314).
  • SA starch asparagine
  • Gene as used herein also includes any regulatory region required for gene function or expression.
  • cvrn genes as used herein refers to any of the genes cvml, cvm2, cvm3, cvm4, cvm5, cvm ⁇ or cvm7 as defined hereinabove.
  • cvmpara genes as used herein refers to any of the genes cvm ⁇ para or cvml par a as defined hereinabove.
  • orf genes as used herein refers to any of the genes orf2, or ⁇ , orf4, or ⁇ , orf 6, or ⁇ , or ⁇ , orflO, orfll, orf 12, orf!3, orfl4, orfl5, orf 16, orfl7, or orf 18 as defined hereinabove.
  • orfpara genes as used herein refers to any of the genes or ⁇ para, or ⁇ para, or ⁇ para or orf ⁇ para as defined hereinabove.
  • Disrupted as used herein means that that the activity of the gene (with regard 5S clavam production) has been reduced or eliminated by, for example, insertional inactivation using an antibiotic resistance gene, preferably apromycin (Paradkar, A.S and Jensen, S.E (1995) supra), or other mutagenesis technique (for example those disclosed in Sambrook et al (1989) supra). Other mutagenesis techniques include insertion of other DNAs (not antibiotic resistance genes), site- directed mutagenesis to either change one or more bases in the gene sequence or insert one or more bases into the sequence of the gene. "Deleted” as used herein means that the gene, or a segment thereof, has been deleted
  • the deletion may be large (for example the complete open reading frame with or without regulatory control regions) or small (for example a single base pair resulting in a frameshift mutation).
  • Reduced as used herein means that the levels of 5S clavam produced by the microorganism of the invention are lower than the levels produced in the corresponding S. clavuligerus st-ain which has not had the relevant open reading frames disrupted or deleted.
  • the corresponding S. clavuligerus is therefore the "parent" strain into which the disrupted or deleted open reading frames were subsequently introduced to generate the microorganism of the invention.
  • At least maintained as used herein means that the level of clavulanic acid produced in the microorganism of the invention is the same or greater than that produced in the corresponding S. clavuligerus strain which has not had the relevant open reading frames disrupted or deleted.
  • the corresponding S. clavuligerus is therefore the "parent" strain into which the disrupted or deleted open reading frames were subsequently introduced to generate the microorganism of the invention.
  • the present invention concerns new processes for making clavulanic acid using newly identified S. clavuligerus genes.
  • a probe derived from orf 4 a fragment of the S. clavuligerus genome has been isolated and has been shown to comprise a number of genes that when disrupted are shown to affect 5S and 5R clavam biosynthesis in S. clavuligerus.
  • Sequence analysis of the fragment has indicated the presence of a gene showing high similarity to orf 4 (hereinafter called orftpar).
  • orftpar a gene showing high similarity to orf 4
  • sequence analysis of the regions flanking the orf4par gene has revealed a new cluster of genes comprising paralogues of genes previously identified in both the clavulanic acid (cas2 cluster) and 5S clavam (casl cluster) gene clusters.
  • the invention provides a S. clavuligerus microorganism comprising DNA corresponding to one or more open reading frames essential for 5S clavam biosynthesis, wherein said open reading frames are disrupted or deleted such that the production of 5S clavams by said S. clavuligerus is reduced and clavulanic acid production is at least maintained, wherein the open reading frames are selected from: a) cvm ⁇ para (SEQ ID NO: 1); b) cvm7para (SEQ TD NO:2); c) cvm ⁇ para and cvm ⁇ (SEQ ID NO:5); or d) cvml par a and cvml (SEQ ID NO: 6).
  • the invention provides a S. clavuligerus microorganism comprising DNA corresponding to one or more open reading frames essential for 5S clavam biosynthesis, wherein said open reading frames are disrupted or deleted such that the production of 5S clavams by said S.
  • clavuligerus is reduced and clavulanic acid production is at least maintained, wherein the open reading frames are selected from: a) cvm ⁇ para and one or more of cvml (SEQ ID NO:7), cvm2 (SEQ ID NO:8), cvm3 (SEQ ID NO:9), cvm4 (SEQ ID NO: 10), cvm5 (SEQ ID NO: 11), cvm ⁇ , cvml or cvmlpara; or b) cvmlpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvm ⁇ , cvml or cvm ⁇ para.
  • the open reading frames are selected from: a) cvm ⁇ para and one or more of cvml (SEQ ID NO:7), cvm2 (SEQ ID NO:8), cvm3 (SEQ ID NO:9), cv
  • cvml is orfupl
  • cvm2 is orfup2
  • cvm3 is orfup3
  • cvm4 is ordwnl
  • cvm5 is orfdwn2
  • cvm ⁇ is orfdwn3.
  • the cvml gene found to be a further 5S clavam specific gene of the 5S clavam (casl) cluster, has been identified during work leading to the present invention and is disclosed hereinbelow.
  • the invention provides isolated polynucleotides comprising the cvm ⁇ para and cvmlpara open reading frames which are used in the preparation of the S. clavuligerus microorganism of the invention.
  • said polynucleotides comprise open reading frames selected from the group consisting of: a) cvm ⁇ para; b) cvmlpara; c) cvm ⁇ para and cvm ⁇ ; d) cvmlpara and cvml; e) cvm ⁇ para and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvm ⁇ , cvml or cvmlpara; or f) cvmlpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvm ⁇ , cvml or cvm ⁇ para.
  • the present invention provides vectors for cloning and manipulating the cvm polynucleotides disclosed herein and which can be used in the preparation of the S. clavuligerus microorganism of the invention. Processes for using these vectors to make the S. clavuligerus microorganism of the invention are also provided.
  • the encoded polypeptides from cvm ⁇ para and cvmlpara are also provided by the invention (SEQ ID NO:3 and SEQ ID NO:4 respectively).
  • the invention further provides a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) or ⁇ para (SEQ ED NO: 12), b) or ⁇ para (SEQ ID NO: 13), c) or ⁇ para (SEQ ID NO: 14), and d) orf ⁇ para (SEQ ID NO: 15).
  • the invention provides a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames comprise one or more of: a) or ⁇ para, b) or ⁇ para, c) or ⁇ para, d) orf ⁇ para in combination with one or more genes involved in clavulanic acid biosynthesis selected from or ⁇ , or ⁇ , or ⁇ , or ⁇ , orf ⁇ , or ⁇ , or ⁇ , or ⁇ , orf 10 (Canadian patent application CA2108113 and Jensen, S.E et al (2000) Antimicrob. Agents Chemother 44:720-6) orf 11, orfl2 (Li, R.N et al (2000) J. Bacteriol 182:4087-95), orf 13, orf 14, orf 15, orf 16, orf 11, or orf 18 (patent application PCT/GB02/04989).
  • open reading frames comprise one or more of: a) or ⁇ para, b) or ⁇ para, c
  • Vectors comprising such polynucleotides are also provided by the present invention together with processes for the use of such vectors to prepare strains of Streptomyces clavuligerus which can be used to produce elevated levels of clavulanic acid.
  • the invention further provides a Streptomyces clavuligerus microorganism comprising a vector comprising a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) or ⁇ para, b) or ⁇ para, c) or ⁇ para, and d) orf ⁇ para.
  • the invention provides a Streptomyces clavuligerus microorganism comprising a vector comprising a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) or ⁇ para, b) or ⁇ para, c) or ⁇ para, d) orf ⁇ para in combination with one or more genes involved in clavulanic acid biosynthesis selected from or ⁇ , or ⁇ , or ⁇ , orf ⁇ , or ⁇ , or ⁇ , or ⁇ , orf 10 (Canadian patent application CA2108113 and Jensen, S.E et al (2000) Antimicrob.
  • the present invention also contemplates a S. clavuligerus micororganism comprising a combination of one or more disrupted or deleted cvm ⁇ para or cvmlpara genes, optionally in combination with other disrupted or deleted 5S genes previously disclosed, together with vectors comprising or ⁇ para, or ⁇ para, or ⁇ para or orf ⁇ para genes, optionally in combination with other clavulanic acid biosynthetic genes (selected from the genes or ⁇ to orf 18) previously disclosed.
  • Polynucleotides of the invention can be isolated by conventional cloning methods, such as PCR or library screening methods, using the sequences disclosed herein and in Mosher et al (1999) supra, WO98/33896, Canadian patent application CA2108113, Jensen, S.E et al (2000) supra), Li, R.N et al (2000) supra and patent application PCT/GB02/04989, as indicated hereinabove. Examples of such cloning methods are described in, for example, Sambrook, J et al (1989) Molecular cloning, a laboratory manual (2nd Ed) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • Polynucleotides comprising individual open reading frames can be isolated and ligated together into vectors in a variety of combinations as defined hereinabove using techniques well know in the art.
  • the choice of vector will depend on the function being carried out, for example cloning, expression, gene inactivation or transfer into S. clavuligerus eg. for gene replacement. In all cases a variety of vectors are available to tlie skilled person and are well l ⁇ iown in the art.
  • vectors are known from Sambrook, J et al (1989) supra for general cloning vectors Hopwood, D.A et al (1985) supra for Streptomyces vectors, Paradkar and Jensen (1995) supra, Mosher et al (1999) supra and WO98/33896 supra for gene disruption and gene replacement vectors and CA2108113 supra for vectors suitable for expression of genes in Str-eptornyces clavuligerus.
  • choice of vector is not limited to just those disclosed in these sources.
  • each open reading frame is suitably positioned relative to a transcriptional promoter, whether this be the native promoter or a heterologous promoter that also functions in the Streptomyces clavuligerus background, or indeed other regulatiry sequence, in such a manner that expression of each open reading frame is optimally achieved.
  • a transcriptional promoter whether this be the native promoter or a heterologous promoter that also functions in the Streptomyces clavuligerus background, or indeed other regulatiry sequence, in such a manner that expression of each open reading frame is optimally achieved.
  • Subsequent manipulation of the polynucleotides, in particular with respect their introduction into the Streptomyces clavuligerus background can be carried out according to standard methods as disclosed in, for example, Hopwood, D.A et al (1985) supra.
  • Disruption of gene sequences, and subsequent gene replacement, can be carried out according to the method of Paradkar, A.S and Jensen, S.E (1995) supra. Deletion of gene sequences can be carried out using well established techniques, for example that disclosed in WO98/33896.
  • Microorganisms of the invention can be prepared from Streptomyces clavuligerus strains including, but not limited to, Streptomyces clavuligerus ATCC 27064 (American Type Culture Collection, Manassas, Virginia, USA), alternatively available as NRRL 3585 (Northern Regional Research Laboratory, Peoria, Illinois, USA).
  • Streptomyces clavuligerus ATCC 27064 American Type Culture Collection, Manassas, Virginia, USA
  • mutant strains of Streptomyces clavuligerus can also be used including those prepared by genetic engineering techniques, or those prepared by strain improvement methods.
  • strains examples include Streptomyces clavuligerus strains 56-1A, 56-3A, 57-2B, 57-1C, 60-1A, 60-2A, 60-3A, 61-1A, 61-2A, 61-3A or 61-4A as disclosed in WO98/33896.
  • the invention relates to a process for improving clavulanic acid production in a suitable microorganism comprising isolating a polynucleotide as described hereinabove, manipulating said polynucleotide, introducing the manipulated polynucleotide into a said suitable microorganism, fermenting said suitable microorganism under conditions whereby clavulanic acid is produced, isolating and purifying clavulanic acid so produced.
  • Manipulation of said polynucleotide may be by means of disrupting or deleting gene sequences in the case of cvmpara genes, optionally together with cvm genes, or by inserting into vectors suitable for expression in the case of orjpara genes, optionally together with orf genes.
  • the suitable microorganism is Streptomyces clavuligerus.
  • Such fermentation, isolation and purification methods are well known in the art, for example the fermentation methods disclosed in UK Patent Specification No. 1,508,977. Methods for using clavulanic acid in the preparation of antibiotic formulations are similarly well l ⁇ iown in the art.
  • Example 1 Materials and Methods In the examples all methods are as described in Sambrook, J. et al supra, Hopwood, D.A. et al. (1985) supra and Kieser, T et al. (2000) Practical Streptomyces Genetics, unless otherwise stated. Transformation methods can also be found in Paradkar, A.S. and Jensen, S.E • (1995) supra.
  • Streptomyces clavuligerus NRRL 3585 was obtained from the Northern Regional
  • E. coli cultures were maintained on LB agar medium and grown in liquid culture in LB medium at 37°C (Sambrook, J et al (19%9)supra). Plasmid-containing cultures were supplemented with appropriate levels of antibiotic.
  • Plasmid D ⁇ A was isolated from potential positive clones and confirmed to carry a 4.3 kb Ncol fragment. A representative clone, pO4H-4, was chosen for further study. The sequencing of the 4.3 kb Ncol fragment was carried out. Analysis of the sequence generated identified three genes, one which had homology to or ⁇ and was called or ⁇ par. The two other genes present were found to have homology with orf ⁇ and cvm ⁇ and were therefore called orf ⁇ par and cvm ⁇ par. This result suggested that this region of D ⁇ A may contain a cluster of genes with paralogues in either the clavulanic acid biosynthetic gene cluster or the cvrn clavam biosynthetic gene cluster.
  • genes or ⁇ par, or ⁇ par and or ⁇ par are adjacent to each other and in the same orientation as their counterparts or ⁇ , or ⁇ and or ⁇ .
  • casl is not downstream of or ⁇ par as cas2 is to or ⁇ in the clavulanic acid pathway but is instead within the clavam cluster (Mosher et al (1999) supra).
  • Another difference between the clavulanic acid cluster and the paralogue arrangement is that orf ⁇ par is end-on-end to or ⁇ par, and so is not in the same orientation as or ⁇ par-4par, whereas orf ⁇ is in the same orientation as orfs2-4 in the clavulanic acid cluster.
  • the gene immediately upstream of orf ⁇ par was found to be a gene that had a paralogue in the clavam and not the clavulanic acid cluster.
  • This gene was called cvm ⁇ par, as it is a paralogue of the cvm ⁇ gene found clustered with casl (Mosher et al (1999) supra).
  • the cvm6 gene encodes an enzyme that is involved in clavam production (orfdwn3 in WO98/33896).
  • cvmlpar Located adjacent to cvm ⁇ par is a new gene called cvmlpar. This gene shows homology to cvml, a gene that is located upstream of cvm3 in the clavam cluster (further described hereinbelow). Upstream of cvml is a new open reading frame, believed to encode a sensor kinase. It encodes an polypeptide of 555 amino acids and shows good similarity to sensor kinase domains of two component response regulator genes.
  • pDA501 is a shuttle vector prepared by fusing the Streptomyces plasmid pIJ486 (Kieser, T et al (2000) supra) to the E.coli plasmid pTZl 8R (Stratagene) by means of their EcoKT and Bam ⁇ TT sites.
  • the resulting construct, 6pDAB was used to transform S.lividans TK24, and finally wild-type S. clavuligerus to thiostrepton (thio at 5 ⁇ g/ml) and apramycin (apr at 20 ⁇ g/ml) resistance.
  • Gene replacement mutants were generated as described by Paradkar and Jensen (1995) supra.
  • the or ⁇ disruptant was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 71%.
  • Mutant 5pDA defective in the or ⁇ par gene was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 12%.
  • the orf ⁇ par gene was disrupted by introduction of a neomycin resistance gene (neo r ) into the Rsr ⁇ l site, approximately midway through the coding region.
  • neo r neomycin resistance gene
  • pO4H-4 was digested with Kpnl to remove or ⁇ par and self ligated to give p5K-6.
  • p5K-6 was digested with Rsr ⁇ l and the neomycin resistance gene, released from pFDNeo-S (Denis and Brzezinski (1992) Gene 111: 115-118.) as a Pstl/EcoRT fragment, was inserted after both fragments had been made blunt by treatment with the Klenow fragment of DNA polymerase I.
  • pNeo5K-6A was obtained which has the neo R gene in the same orientation as the orf ⁇ par gene.
  • a shuttle vector called pNeo5K-6Atsr#14 was constructed by inserting pIJ486 as a
  • Mutant 6-1 A defective in the orf ⁇ gene was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 57%. From these results it can be concluded that orf ⁇ is required for efficient production of clavulanic acid as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation Analysis of orf ⁇ par mutants Mutant 14-2B(2) defective in the orf ⁇ par gene was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 27%.
  • a 1.7 Kb Sail fragment containing cvm ⁇ par was released from pO4H-4 and ligated into pUC118 at the Sail site.
  • the resulting plasmid was digested with iscoNI to release a 140 bp fragment internal to cvm ⁇ par.
  • the neomycin resistance gene from pFDNeo-S released as an EcoKTIPstl fragment, was ligated into cvm ⁇ par after both fragments had been made blunt by treatment with the Klenow fragment of DNA polymerase I.
  • the neo R marker was inserted in tlie same orientation as cvm ⁇ par.
  • neomycin containing Sail fragment was released with EcoKl and inserted into tlie shuttle vector pUWL-KS (Weimeier, U.F (1995) Gene 165: 149-150.) at the Ec ⁇ KT site.
  • the construct was named pNeoSall.7U.
  • the plasmid pNeoSall .7U was used to transform S.lividans TK24, and finally S. clavuligerus wild type.
  • the resulting cvm ⁇ par: :neo transformants were selected on MYM medium with 50 ⁇ g/ml neomycin and 5 ⁇ g/ml thiostrepton and then subjected to two rounds of sporulation under non- selective conditions to give double cross-over mutants.
  • the construct pNeoSall .7U isolated from S.lividans TK24 was also used to transform the cvm ⁇ mutant 56-3 A, where the apr 11 cassette was inserted into cvm ⁇ in the same orientation as the gene.
  • Transformants were grown on MYM medium with 50 ⁇ g/ml neomycin and 5 ⁇ g/ml thiostrepton. The mutants were put through two rounds of sporulation under non- selective conditions as described above and double cross-over mutants were isolated.
  • mutants 56-1 A, 56-3A, 57-1C and 57-2B defective in the cvm6 gene produced elevated levels of clavulanic acid (125-141% of the control strain) and greatly reduced levels of clavam-2-carboxylate and 2- hydroxymethylclavam when cultured in Soy medium.
  • the plasmid p5.7£coRI ref (pJOE based hyg) was used as the disruption template for or ⁇ par.
  • the insert in this plasmid is approximately 5.7kb and includes part of cvm ⁇ par, all of orf ⁇ par, or ⁇ par, or ⁇ par and part of or ⁇ par all carried within the plasmid pJOE829 (Kieser, T et al. (2000); Aidoo et al. (1994) Gene. 147:41-6).
  • the disruption vector was constructed by ligation of a thiostrepton resistance cassette (Aidoo et al. supra) into Fsel digested p5.7i?eoRI.
  • a unique Fsel site is located within the insert 507 bp from the start of or ⁇ par.
  • the correct construct was obtained and used to sequentially transform S.lividans TK24 and then S. clavuligerus wild type. Primary transformants were selected on thiostrepton (5 ⁇ g/ml) and hygromycin (25 ⁇ g/ml). The mutants were put through two rounds of sporulation under non- selective conditions as described above and putative double cross-over mutants were isolated.
  • the or ⁇ par disruption cassette described in section 6.2 was isolated from S.lividans TK24 and used to transform or 3::apra mutants. Transformants were selected on MYM medium containing thiostrepton (5 ⁇ g/ml) and hygromycin (25 ⁇ g/ml). The mutants were put through two rounds of sporulation without selection and double crossover mutants isolated as previously described.
  • Mutants Ap3-1, Ap3-2 and Ap3-3 were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulations in clavulanic acid were reduced by 31-71%.
  • Mutants 3A-1 and 3A-2 were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulations in clavulanic acid were reduced by 9%.
  • Mutants disrupted in or ⁇ were originally made as described in United States Patent No. 6,332,106. These original or ⁇ mutants were subjected to a second round of gene replacement to remove the apramycin resistance gene and replace it with a simple frameshift mutation.
  • the plasmid construct used to create the original or ⁇ mutant consisted of a 2.1 kb EcoRT/BglR fragment of S. clavuligerus DNA carried on a pUCl 19/pIJ486 shuttle vector, with the or ⁇ gene disrupted by insertion of an apramycin resistance gene cassette into a centrally located Notl site (United States Patent No. 6,332,106).
  • the disruption plasmid construct used in the second round of mutation was derived from the original disruption plasmid by digestion with Notl to release the apramycin resistance gene cassette, treatment with the Klenow fragment of D ⁇ A polymerase I to fill in the overhanging ends, and then re- ligation to circularize the plasmid.
  • the resulting plasmid construct carries the entire or ⁇ gene but with a frameshift introduced at the location of tlie destroyed Ncol site.
  • the construct was used to sequentially transform S.lividans TK24 and then the original S. clavuligerus or ⁇ mutant. Primary transformants were selected on thiostrepton (5 ⁇ g/ml) and then subjected to two rounds of sporulation under non-selective conditions. Putative double cross-over mutants were identified based on their loss of apramycin resistance .
  • KTA15 5 '-CGGGGCCGGGCATGGTGAACTCGTCCTCCACGGTGGTCATGTAGGC- TGGAGCTGCTT-3', designed to disrupt the or ⁇ par gene by insertion of an apramycin resistance gene, were synthesized.
  • the or ⁇ par disruption cassette was generated by PCR using these two primers with the plasmid pIJ773 as template. PCR conditions used wereas described in the user instructions except that no dimethylsulfoxide was used.
  • the or ⁇ par disruption cassette was then introduced by electrotransformation into E. coli BW25113/pIJ790 which had been previously transformed with the or ⁇ par bearing cosmid 14E10 (described hereinabove).
  • Cosmid DNA was isolated from transformants after overnight growth at 37°C to promote loss of the pIJ790 plasmid and analyzed to confirm that the or ⁇ par gene had been disrupted, or ⁇ par disrupted cosmid DNA was then transferred into wild type S. clavuligerus by conjugation. Conjugation was carried out as described by Kieser, T et al (2000) supra except that AS-1 medium (Baltz, R. H. Genetic recombination by protoplast fusion in Str'eptomyces. Dev. h d. Microbiol 21 (1980) 43-54) supplemented with apramycin at 50 ⁇ g/ml was used for recovery of transconjugants. Apramycin resistant S. clavuligerus transconjugants were subjected to one round of sporulation under non-selective conditions in order to generate gene replacement mutants as described by Paradkar and Jensen (1995) supra.
  • the PCR-based targeting procedure used to generate the orf2par mutants was also used to generate orf2/orf2par double mutants.
  • the orf2par disrupted cosmid DNA was conjugated into the orf2 mutants described above (section 7.1) rather than into the wild type strain.
  • Apramycin resistant S. clavuligerus transconjugants were subjected to one round of sporulation under non-selective conditions in order to obtain unigenomic mutant spores that had undergone gene replacement as previously described.
  • Mutants defective in the or ⁇ gene were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulations in clavulanic acid were reduced by 95-98% (Jensen et al (2000) supra.
  • Mutants defective in the or ⁇ par gene were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 10-30%.
  • the cosmid 10D7 (described in WO98/33896) was digested with the restriction endonuclease Sacl. From this digestion a 6.8 kilobase DNA fragment containing casl and cvml was isolated and cloned into a pUCl 19 based plasmid.
  • the resultant plasmid pCEC019 was used as a template to generate sequence information which allowed completion of the partial cvm3 gene reported in WO98/33896. h addition, the sequence information showed the presence of another open reading frame, cvml, which was incomplete in this fragment.
  • Figure 1 Diagram of the paralogue cluster. The orientation of transcription is shown for each gene (direction of arrow)
  • Figure 2. Orientation of cvml in relation to published cvm cluster (WO98/33896).
  • Figure 3 Annotated seqence of the paralogue cluster
  • SEQ ID NO: 16 paralogue cluster SEQ ID NO: 17 extended cvm cluster (underlined sequence denotes new sequence over that disclosed in WO98/33896
  • VSTAVSPRYAQPATFMRLRHRPDPIGHDVVWGAPYDGGTSYRPGARFAPRAIRHESSLIHGVGIDRGPGVFDRID VVDGGDIDLSPFSMDLAMDTATVALTRLLERNDAFL LGGDHSLSLAALRAVHARHGRVAVLHLDAHSDTNPPVYG GTYHHGTPFRWAIEEGLVDPERLVQVGIRGHNPRPDSLDYARGHGVSIVTAADFTRRSPRGIAEQIRRTVGGLPLY VSVDIDVVDPAYAPGTGTPAPGGLSSREVLT LDVVGQLRPVGFDVVEVSPAYDPSGITSLLAAEIGAELLYQYAR ATTSPASAPVDSPLPPGAAADDAENAENAVDAVDAESAVDFAGQRWG.

Abstract

New processes for improving the manufacture of clavams e.g. clavulanic acid. Novel DNA sequences and new microorganisms capable of producing increased amounts of clavulanic acid are also disclosed.

Description

New Process
The present invention relates to new processes for improving the manufacture of clavams e.g. clavulanic acid. The present invention also provides novel DNA sequences and new microorganisms capable of producing increased amounts of clavulanic acid.
Microorganisms, in particular Streptomyces sp. produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins, polyketides, cephamycins, tunicamycin, holomycin and penicillins. There is considerable interest in being able to manipulate the absolute and relative amounts of these antibiotics produced by the microorganism and accordingly there have been a large number of studies investigating the metabolic and genetic mechanisms of the biosynthetic pathways (Demain, A.L. (1990) "Biosynthesis and regulation of β-lactam antibiotics." in "50 years of Penicillin applications, history and trends").
Streptomyces clavuligerus produces two major groups of antibiotics; one being the cephamycins, cephalosporins and penicillins (Demain, A.L. (1990) supra) and the other comprising clavams. Clavams can be arbitrarily divided into two groups, 5S and 5R clavams, dependent on their ring stereochemistry. The commercially important clavam clavulanic acid, a component of the antibiotic Augmentin (trade mark of GlaxoSmithKline), is a 5R clavam. Examples of 5S clavams are clavam-2-carboxylate (C-2-C), 2-hydroxymethyl clavam (2HMC) and alanylclavam (Brown et al. (1979) J. Chem. Soc. Chem. pp282-283). Genes encoding biosynthetic enzymes and regulatory proteins for clavulanic acid production have been located in a cluster next to the genes involved in cephamycin C production and make up a supercluster of antibiotic related genes within the S. clavuligerus genome (Alexander et al. (1998) J.Bacteriol. 180:4068-79). For example the genes encoding the enzymes involved in clavaminic acid production, a clavulanic acid precursor, which include orβ (ceaS) (Khaleeli et al. (1999) J. Am. Chem. Soc. 121:9223-9224), orβ (bis) (Bachmann and Townsend (1998) Chem. Commun.:2325-2326), orf4 (pah) (Wu et al. (1995) J. Bacteriol. 177:3714-3720), orf5 (cas2) (Marsh et al. (1992) Biochemistry. 31 : 12648-57) and perhaps orfό (Kershaw et al (2002) Eur. J. Biochem. 269,2052-2059) are all located within the clavulanic acid cluster. Disruptions in orfs2-6 cause a complete loss of clavulanic acid production when mutant cultures are grown on starch asparagine medium (Aidoo, K.A. et al. (1993) p219-236 In. V.P. Gullo, J.C. Hunter-Cevera, R. Cooper and R. K. Johnson (ed.), Developments in Industrial Microbiology series, vol.33 Society for Industrial Microbiology, Fredericksburg, Va.). However this loss is conditional upon the growth media used for when mutants are grown on Soy medium (Salowe et al. (1990) Biochemistry 29: 6499-6508) clavulanic acid production is partially restored (Jensen et al. (2002) Antimicrob. Agents and Chemother. 44: 720-726). This phenomenon could suggest that other genes present in the S. clavuligerus genome could compensate in some way for the loss of the activity of these genes under certain conditions. Alternatively it could be that the Soy media contains very small amounts one or more of the metabolites produced by the orfs 2-6 allowing strains disrupted in these genes to make small amounts of clavulanic acid. Marsh et al. (1992) supra has reported that S. clavuligerus contains two copies of the cas gene (casl and cas2). casl is not associated with the clavulanic acid gene cluster and has a high homology to casl. Disruption of casl decreases clavulanic acid production by 35% when cultures are grown on Soy medium and eliminates production entirely when cultures are grown on starch asparagine (SA) medium (Paradkar and Jensen 1995 J.Bact 177: 1307- 1314). The disruption of the casl gene results in mutants which produce near wild type levels of clavulanic acid on SA medium, but produce 31-73% less clavulanic acid when grown on Soy medium than the wild type (Mosher et al (1999) Antimicrob. Agents and Chemother. 43: 1215-1224). It is also reported that in mutant strains where both the casl and cas2 genes have been disrupted no clavulanic acid is produced under any of the fermentation conditions tested. hiterestingly when the genes surrounding casl were sequenced, no additional genes involved in clavulanic acid production were found but instead six novel genes involved in 5S clavam biosynthesis (named cvml to 6) were identified. (Mosher et al (1999) supra). Further work on these 5S clavam-specifϊc genes showed that disruption of the genes, using genetic engineering methodologies, leads to improvements in the levels of clavulanic acid made by the mutant strains and also dramatic reductions in the levels of 5S clavam production
(WO98/33896). This reduction in 5S clavam production, in particular the 5S clavam clavam- 2-carboxylate, is especially important in the commerical production of clavulanic acid because some 5S clavams are known to be toxic and for this reason the levels are tightly controlled within the British and US Pharmacopoeias. Despite these advances in the understanding of clavulanic acid biosynthesis it is still a highly desirable goal in the pharamceutical industry to continue to improve production methods for clavulanic acid, both for reasons of cost and for reasons of safety.
The following definitions are provided to facilitate understanding of certain terms used frequently herein: "Gene" as used herein also includes any regulatory region required for gene function or expression.
"cvrn" genes as used herein refers to any of the genes cvml, cvm2, cvm3, cvm4, cvm5, cvmό or cvm7 as defined hereinabove.
" cvmpara" genes as used herein refers to any of the genes cvmόpara or cvml par a as defined hereinabove.
" orf genes as used herein refers to any of the genes orf2, orβ, orf4, orβ, orf 6, orβ, orβ, orβ, orflO, orfll, orf 12, orf!3, orfl4, orfl5, orf 16, orfl7, or orf 18 as defined hereinabove. "orfpara" genes as used herein refers to any of the genes orβpara, orβpara, orβpara or orfόpara as defined hereinabove.
"Disrupted" as used herein means that that the activity of the gene (with regard 5S clavam production) has been reduced or eliminated by, for example, insertional inactivation using an antibiotic resistance gene, preferably apromycin (Paradkar, A.S and Jensen, S.E (1995) supra), or other mutagenesis technique (for example those disclosed in Sambrook et al (1989) supra). Other mutagenesis techniques include insertion of other DNAs (not antibiotic resistance genes), site- directed mutagenesis to either change one or more bases in the gene sequence or insert one or more bases into the sequence of the gene. "Deleted" as used herein means that the gene, or a segment thereof, has been deleted
(removed) from a larger polynucleotide which, before the deletion was perfoπned, included said gene or segment thereof. When the polynucleotide bearing the deletion is introduced into the genome of the microorganism by means of gene replacement technology (Paradkar and Jensen (1995) supra) the activity of the gene or protein encoded thereby is eliminated or reduced such that the levels of 5S clavam produced by the microorganism are reduced. The deletion may be large (for example the complete open reading frame with or without regulatory control regions) or small (for example a single base pair resulting in a frameshift mutation).
"Reduced" as used herein means that the levels of 5S clavam produced by the microorganism of the invention are lower than the levels produced in the corresponding S. clavuligerus st-ain which has not had the relevant open reading frames disrupted or deleted. The corresponding S. clavuligerus is therefore the "parent" strain into which the disrupted or deleted open reading frames were subsequently introduced to generate the microorganism of the invention.
"At least maintained" as used herein means that the level of clavulanic acid produced in the microorganism of the invention is the same or greater than that produced in the corresponding S. clavuligerus strain which has not had the relevant open reading frames disrupted or deleted. The corresponding S. clavuligerus is therefore the "parent" strain into which the disrupted or deleted open reading frames were subsequently introduced to generate the microorganism of the invention.
The present invention concerns new processes for making clavulanic acid using newly identified S. clavuligerus genes. Using a probe derived from orf 4 a fragment of the S. clavuligerus genome has been isolated and has been shown to comprise a number of genes that when disrupted are shown to affect 5S and 5R clavam biosynthesis in S. clavuligerus. Sequence analysis of the fragment has indicated the presence of a gene showing high similarity to orf 4 (hereinafter called orftpar). However surprisingly further sequence analysis of the regions flanking the orf4par gene has revealed a new cluster of genes comprising paralogues of genes previously identified in both the clavulanic acid (cas2 cluster) and 5S clavam (casl cluster) gene clusters.
Accordingly the invention provides a S. clavuligerus microorganism comprising DNA corresponding to one or more open reading frames essential for 5S clavam biosynthesis, wherein said open reading frames are disrupted or deleted such that the production of 5S clavams by said S. clavuligerus is reduced and clavulanic acid production is at least maintained, wherein the open reading frames are selected from: a) cvmόpara (SEQ ID NO: 1); b) cvm7para (SEQ TD NO:2); c) cvmόpara and cvmό (SEQ ID NO:5); or d) cvml par a and cvml (SEQ ID NO: 6).
In a second aspect the invention provides a S. clavuligerus microorganism comprising DNA corresponding to one or more open reading frames essential for 5S clavam biosynthesis, wherein said open reading frames are disrupted or deleted such that the production of 5S clavams by said S. clavuligerus is reduced and clavulanic acid production is at least maintained, wherein the open reading frames are selected from: a) cvmόpara and one or more of cvml (SEQ ID NO:7), cvm2 (SEQ ID NO:8), cvm3 (SEQ ID NO:9), cvm4 (SEQ ID NO: 10), cvm5 (SEQ ID NO: 11), cvmό, cvml or cvmlpara; or b) cvmlpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvmό, cvml or cvmόpara.
The genes cvml, cvrn2, cvm3, cvm4, cvm5 and cvmό are disclosed in Mosher et al (1999) supra and WO98/33896 (cvml is orfupl, cvm2 is orfup2, cvm3 is orfup3, cvm4 is ordwnl, cvm5 is orfdwn2 and cvmό is orfdwn3. The cvml gene, found to be a further 5S clavam specific gene of the 5S clavam (casl) cluster, has been identified during work leading to the present invention and is disclosed hereinbelow.
In a further aspect the invention provides isolated polynucleotides comprising the cvmόpara and cvmlpara open reading frames which are used in the preparation of the S. clavuligerus microorganism of the invention. Preferably said polynucleotides comprise open reading frames selected from the group consisting of: a) cvmόpara; b) cvmlpara; c) cvmόpara and cvmό; d) cvmlpara and cvml; e) cvmόpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvmό, cvml or cvmlpara; or f) cvmlpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvmό, cvml or cvmόpara.
In another aspect the present invention provides vectors for cloning and manipulating the cvm polynucleotides disclosed herein and which can be used in the preparation of the S. clavuligerus microorganism of the invention. Processes for using these vectors to make the S. clavuligerus microorganism of the invention are also provided.
The encoded polypeptides from cvmόpara and cvmlpara are also provided by the invention (SEQ ID NO:3 and SEQ ID NO:4 respectively). The invention further provides a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) orβpara (SEQ ED NO: 12), b) orβpara (SEQ ID NO: 13), c) orβpara (SEQ ID NO: 14), and d) orfόpara (SEQ ID NO: 15).
In a further aspect the invention provides a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames comprise one or more of: a) orβpara, b) orβpara, c) orβpara, d) orfόpara in combination with one or more genes involved in clavulanic acid biosynthesis selected from orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβ, orf 10 (Canadian patent application CA2108113 and Jensen, S.E et al (2000) Antimicrob. Agents Chemother 44:720-6) orf 11, orfl2 (Li, R.N et al (2000) J. Bacteriol 182:4087-95), orf 13, orf 14, orf 15, orf 16, orf 11, or orf 18 (patent application PCT/GB02/04989).
Vectors comprising such polynucleotides are also provided by the present invention together with processes for the use of such vectors to prepare strains of Streptomyces clavuligerus which can be used to produce elevated levels of clavulanic acid.
Strains of Str-eptomyces clavuligerus so produced and methods for using them to produce clavulanic acid by fermentation are also provided.
Thus the invention further provides a Streptomyces clavuligerus microorganism comprising a vector comprising a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) orβpara, b) orβpara, c) orβpara, and d) orfόpara. In a further aspect the invention provides a Streptomyces clavuligerus microorganism comprising a vector comprising a polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) orβpara, b) orβpara, c) orβpara, d) orfόpara in combination with one or more genes involved in clavulanic acid biosynthesis selected from orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβ, orf 10 (Canadian patent application CA2108113 and Jensen, S.E et al (2000) Antimicrob. Agents Chemother 44:720-6) or/11, orfl2 (Li, R.N et al (2000) J. Bacteriol 182:4087-95), orf 13, orf 14, orf 15, orf 16, orf 11, or orf 18 (patent application PCT/GB02/04989).
The present invention also contemplates a S. clavuligerus micororganism comprising a combination of one or more disrupted or deleted cvmόpara or cvmlpara genes, optionally in combination with other disrupted or deleted 5S genes previously disclosed, together with vectors comprising orβpara, orβpara, orβpara or orfόpara genes, optionally in combination with other clavulanic acid biosynthetic genes (selected from the genes orβ to orf 18) previously disclosed.
Polynucleotides of the invention can be isolated by conventional cloning methods, such as PCR or library screening methods, using the sequences disclosed herein and in Mosher et al (1999) supra, WO98/33896, Canadian patent application CA2108113, Jensen, S.E et al (2000) supra), Li, R.N et al (2000) supra and patent application PCT/GB02/04989, as indicated hereinabove. Examples of such cloning methods are described in, for example, Sambrook, J et al (1989) Molecular cloning, a laboratory manual (2nd Ed) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Polynucleotides comprising individual open reading frames can be isolated and ligated together into vectors in a variety of combinations as defined hereinabove using techniques well know in the art. The choice of vector will depend on the function being carried out, for example cloning, expression, gene inactivation or transfer into S. clavuligerus eg. for gene replacement. In all cases a variety of vectors are available to tlie skilled person and are well lαiown in the art. For example such vectors are known from Sambrook, J et al (1989) supra for general cloning vectors Hopwood, D.A et al (1985) supra for Streptomyces vectors, Paradkar and Jensen (1995) supra, Mosher et al (1999) supra and WO98/33896 supra for gene disruption and gene replacement vectors and CA2108113 supra for vectors suitable for expression of genes in Str-eptornyces clavuligerus. However the choice of vector is not limited to just those disclosed in these sources. Further, in the case of the gene combinations involving the orβpara, orβpara, orβpara, orβpara and orfόpara genes the skilled artisan would be able to design suitable DNA constructs to ensure that each open reading frame is suitably positioned relative to a transcriptional promoter, whether this be the native promoter or a heterologous promoter that also functions in the Streptomyces clavuligerus background, or indeed other regulatiry sequence, in such a manner that expression of each open reading frame is optimally achieved. Subsequent manipulation of the polynucleotides, in particular with respect their introduction into the Streptomyces clavuligerus background, can be carried out according to standard methods as disclosed in, for example, Hopwood, D.A et al (1985) supra. Disruption of gene sequences, and subsequent gene replacement, can be carried out according to the method of Paradkar, A.S and Jensen, S.E (1995) supra. Deletion of gene sequences can be carried out using well established techniques, for example that disclosed in WO98/33896.
Microorganisms of the invention can be prepared from Streptomyces clavuligerus strains including, but not limited to, Streptomyces clavuligerus ATCC 27064 (American Type Culture Collection, Manassas, Virginia, USA), alternatively available as NRRL 3585 (Northern Regional Research Laboratory, Peoria, Illinois, USA). For example mutant strains of Streptomyces clavuligerus can also be used including those prepared by genetic engineering techniques, or those prepared by strain improvement methods. Examples of such strains include Streptomyces clavuligerus strains 56-1A, 56-3A, 57-2B, 57-1C, 60-1A, 60-2A, 60-3A, 61-1A, 61-2A, 61-3A or 61-4A as disclosed in WO98/33896.
Thus in another aspect the invention relates to a process for improving clavulanic acid production in a suitable microorganism comprising isolating a polynucleotide as described hereinabove, manipulating said polynucleotide, introducing the manipulated polynucleotide into a said suitable microorganism, fermenting said suitable microorganism under conditions whereby clavulanic acid is produced, isolating and purifying clavulanic acid so produced. Manipulation of said polynucleotide may be by means of disrupting or deleting gene sequences in the case of cvmpara genes, optionally together with cvm genes, or by inserting into vectors suitable for expression in the case of orjpara genes, optionally together with orf genes. Preferably the suitable microorganism is Streptomyces clavuligerus. Such fermentation, isolation and purification methods are well known in the art, for example the fermentation methods disclosed in UK Patent Specification No. 1,508,977. Methods for using clavulanic acid in the preparation of antibiotic formulations are similarly well lαiown in the art.
Examples
Example 1 - Materials and Methods In the examples all methods are as described in Sambrook, J. et al supra, Hopwood, D.A. et al. (1985) supra and Kieser, T et al. (2000) Practical Streptomyces Genetics, unless otherwise stated. Transformation methods can also be found in Paradkar, A.S. and Jensen, S.E • (1995) supra.
1.1 Bacterial strains, media and culture conditions. Streptomyces clavuligerus NRRL 3585 was obtained from the Northern Regional
Research Laboratory (Peoria, IL). S. clavuligerus was maintained on either MYM agar (Stuttard, C. (1982) J. Gen. Microbiol. 128: 115-121) or ISP Medium #4 agar plates (Difco, Detroit, MI).
Cultures for the isolation of chromosomal DNA were grown on a 2:3 mixture of trypticase soy broth and YEME as described by Alexander et al.(1998) J.Bact. 180:4068-79. Cultures for analysis of the production of clavulanic acid and other clavam metabolites were grown on Soy medium (European Patent 0349 121) unless otherwise stated. All liquid cultures were grown at 26°C on a rotary shaker at 250 rpm.
Manipulation of DNA in Escherichia coli was done using strain XL-1 Blue (Stratagene, La Jolla, CA). E. coli cultures were maintained on LB agar medium and grown in liquid culture in LB medium at 37°C (Sambrook, J et al (19%9)supra). Plasmid-containing cultures were supplemented with appropriate levels of antibiotic.
1.2 DNA manipulations. Standard DNA manipulations such as plasmid isolation, restriction endonuclease digestion, generation of blunt-ended fragments, ligation, 32P labelling of DNA probes by nick translation and E. coli transformation were carried out as described in Sambrook J et al (1989) supra). Plasmid and genomic DNA isolation from Streptomyces spp. was conducted as described in Kieser, T et al (2000) supra. Construction of a library of S. clavuligerus genomic DNA fragments in the cosmid pWE15 was carried out according to the manufacturer's instructions (Stratagene).
Southern analysis of S. clavuligerus DNA fragments was conducted at high stringency as described by Sambrook, J et al (1989) supra. Hybridization membranes were washed twice for 30 min at 2xSSC/0.1% SDS and once for 30 min at O.lxSSC/0.1% SDS, all at 65°C.
Example 2 - Preparation of the paralogue cluster DNA fragment
2.1 Cloning and nucleotide sequencing of the orf4 paralogue
A strong and a very weak hybridization signal was consistently observed on Southern blots of Ncol-digested S. clavuligerus chromosomal D A when probed with the orβ gene (CA2108113). The strong signal corresponded to the orβ gene, but the identity of the gene that gave rise to the very weak signal was unknown. Therefore it was decided to clone this gene. To this end, Ncol fragments from S. clavuligerus D A of approximately 4-5kb in size were ligated into Ncol digested pUC120 (Vieira, J and J Messing (1987) Methods Enzymol. 153, 3-11) and screened using a colony blot hybridisation method and employing the orβ gene as a probe. Plasmid DΝA was isolated from potential positive clones and confirmed to carry a 4.3 kb Ncol fragment. A representative clone, pO4H-4, was chosen for further study. The sequencing of the 4.3 kb Ncol fragment was carried out. Analysis of the sequence generated identified three genes, one which had homology to orβ and was called orβpar. The two other genes present were found to have homology with orfό and cvmό and were therefore called orfόpar and cvmόpar. This result suggested that this region of DΝA may contain a cluster of genes with paralogues in either the clavulanic acid biosynthetic gene cluster or the cvrn clavam biosynthetic gene cluster.
2.2 Sequencing of DΝA flanking the 4.3 kb Ncol fragment containing orf4par
Sequence analysis of DΝA flanking the 4.3 kb Ncol fragment containing or 4par was achieved by identifying 2 cosmid clones containing the orβpar gene. The two cosmid clones containing orf4par,14E10 and 6G9, were isolated from a S. clavuligerus pWE15 (Promega, Madison, WI) cosmid bank that had been probed with a 0.46Kb SaR fragment that is internal to the orβpar gene. These cosmids have been partially mapped using a series of digestions and Southern hybridization experiments (In. Nucleic acid techniques in bacterial systematics. Ed. Stackebrandt, E and Goodfellow, M (1991) John Wiley and Sons, p205-248). Digestion of both cosmids with iscoRI, Kpή and Nrøl suggest that the insert size of 14E10 is approximately 45 kb and 6G9 is approximately 40 kb. These two cosmid inserts have about 20 kb of overlapping DΝA and provided DΝA for sequence analysis of regions upstream and downstream of the 4.3 kb Ncol fragment containing orβpar. DΝA sequence information was generated essentially as described in CA2108113. The
DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia, Baie d'Urfe, Quebec, Canada) was used. Approximately 13.3 kilobases of contiguous DΝA sequence was generated. The nucleotide sequence of the S. clavuligerus chromosomal DΝA generated in these experiments is shown in SEQ ID No: 16. A number of open reading frames were identified which displayed significant homology with the previously described orβ, orβ, or , and orfό (CA2108113). These genes have been located within the genome in relation to each other, and are found to be nearly in the same organisation as that of the genes within the clavulanic acid cluster. The genes orβpar, orβpar and orβpar are adjacent to each other and in the same orientation as their counterparts orβ, orβ and orβ. However casl is not downstream of orβpar as cas2 is to orβ in the clavulanic acid pathway but is instead within the clavam cluster (Mosher et al (1999) supra). Another difference between the clavulanic acid cluster and the paralogue arrangement is that orfόpar is end-on-end to orβpar, and so is not in the same orientation as orβpar-4par, whereas orfό is in the same orientation as orfs2-4 in the clavulanic acid cluster. Suprisingly the gene immediately upstream of orfόpar, was found to be a gene that had a paralogue in the clavam and not the clavulanic acid cluster. This gene was called cvmόpar, as it is a paralogue of the cvmό gene found clustered with casl (Mosher et al (1999) supra). The cvm6 gene encodes an enzyme that is involved in clavam production (orfdwn3 in WO98/33896).
Located adjacent to cvmόpar is a new gene called cvmlpar. This gene shows homology to cvml, a gene that is located upstream of cvm3 in the clavam cluster (further described hereinbelow). Upstream of cvml is a new open reading frame, believed to encode a sensor kinase. It encodes an polypeptide of 555 amino acids and shows good similarity to sensor kinase domains of two component response regulator genes.
2.3 Functional analysis of the open reading frames Computer analysis of the DNA sequence shown in SEQ ID No.16 predicts the presence of 7 open reading frames. A description of each gene is shown in Table 1.
Table 1
To assess the possible roles of these ORFs in the biosynthesis of clavulanic acid and/or clavams produced by S. clavuligerus, insertional inactivation mutants were created by gene replacement essentially as described by Paradkar and Jensen (1995) supra. However, in order to definitively define the phenotype of these disruptions, it was considered important to disrupt orβpar, orβpar, orfόpar and cvmόpαr not only in wild type S. clavuligerus, but also in strains of S. clavuligerus that were already defective in the expression of orβ, orβ, orfό, and cvmό respectively. The orβ, 4 and 6 mutants were made as described in United States Patent No. 6,332,106 and the cvmό mutant made as described in WO98/33896.
Example 3 - Analysis orf 4, and orf4par
3.1 Construction of orβ mutants
Mutants disrupted in orβ (pah) were made as described in United States Patent No. 6,332,106.
3.2 Construction of orβpar mutants p04H-4 (4.3kb Ncol fragment cloned into the Ncol site of pUC120 (Vieira and Messing 1987 supra) was digested with Kpnl (one site in the cloned fragment and one site in the vector) and religated to reduce the size of the orβpar-beaxm' g DΝA insert to 1.7kb thereby generating the plasmid p4K-l. The orβpαr gene within p4K-l was disrupted by digestion at its centrally located .EcoΝI site and insertion of the apramycin (αpr) resistance gene cassette from pUC120apr (Trepanier et al. (2002) Microbiology 148: 643-656) after both fragments had been made blunt by treatment with the Klenow fragment of DΝA polymerase I. The Kpή /Ncol insert carrying the disrupted orβpαr gene was then inserted into the iscoRI site of pDA501 after blunting the ends of both insert and vector. pDA501 is a shuttle vector prepared by fusing the Streptomyces plasmid pIJ486 (Kieser, T et al (2000) supra) to the E.coli plasmid pTZl 8R (Stratagene) by means of their EcoKT and BamϊTT sites. The resulting construct, 6pDAB, was used to transform S.lividans TK24, and finally wild-type S. clavuligerus to thiostrepton (thio at 5μg/ml) and apramycin (apr at 20μg/ml) resistance. Gene replacement mutants were generated as described by Paradkar and Jensen (1995) supra.
3.3 Construction of orβ/orβpar mutants An approach was undertaken to generate the double mutant by transforming protoplasts of the orβpar (aprr) mutant with the orβ (thio1) disruption construct (Aidoo et al. (1994) Gene. 147:41-6). Protoplast preparations from orβpar mutants, were transformed with the orβ disruption construct isolated from S.lividans. Transformants were selected on thiostrepton at 5μg/ml and hygromycin (hyg) at 50μg/ml. Primary transformants were put through two rounds of sporulation under non- selective conditions in order to generate gene replacement mutants as described by Paradkar and Jensen (1995) supra.
3.4 Fermentation analysis of orβ, orβpar and orβ/orf4par mutants
To test the effect of disrupting orβ, orβpar and orβ/4par on clavulanic acid biosynthesis, spores from each isolate were inoculated into 20ml of seed medium (European patent 0 349 121) and grown for 2 days at 26°C with shaking. 1ml of the seed culture was then inoculated into a final stage Soy medium (European Patent 0349 121) and grown at 26°C for up to 3 days with shaking. Samples of final stage broth were withdrawn after three days growth and assayed for clavulanic acid productivity by HPLC (Mosher et al (1999) supra) and/ or using an imidazole derivatised colorimetric assay (Bird, A.E. et al (1982) Analyst, 107: 1241-1245 and Foulston, M. and Reading, C. (1982) Antimicrob. Agents Chemother., 22:753-762). Fermentation analysis of orβ disruptant
The orβ disruptant was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 71%.
From these results it can be concluded that orβ is required for efficient production of clavulanic acid as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation analysis of orβpar disruptant
Mutant 5pDA defective in the orβpar gene was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 12%.
From these results it can be concluded that, like orβ, orβpar contributes to clavulanic acid biosynthesis as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation analysis of orf4/orβpar disruptants When mutants A4-A1 and 3 A3 -A3, defective in both copies of the orβ genes were grown in Soy medium production of clavulanic acid could not be detected.
From these results it can be concluded that under the conditions tested, both genes, orβ and orβpar, contribute to clavulanic acid biosynthesis as the double disruption, results in a mutant unable to make clavulanic acid.
3.5 Southern Analysis
The orβ, orβpar and orf4/4par mutants were further characterised by Southern analysis. The results confirmed that in these mutants the chromosomal copies of the relevant genes had been disrupted as expected.
Example 4 - Analysis of orfό and orfόpar
4.1 Construction of orβ mutants orfό mutants were made as described in United States Patent No. 6,332,106
4.2 Construction of orfόpar mutants
The orfόpar gene was disrupted by introduction of a neomycin resistance gene (neor) into the Rsrϊl site, approximately midway through the coding region. In order to achieve this pO4H-4 was digested with Kpnl to remove orβpar and self ligated to give p5K-6. p5K-6 was digested with Rsrϊl and the neomycin resistance gene, released from pFDNeo-S (Denis and Brzezinski (1992) Gene 111: 115-118.) as a Pstl/EcoRT fragment, was inserted after both fragments had been made blunt by treatment with the Klenow fragment of DNA polymerase I. The construct pNeo5K-6A was obtained which has the neoR gene in the same orientation as the orfόpar gene. A shuttle vector called pNeo5K-6Atsr#14 was constructed by inserting pIJ486 as a
6.2 Kb fragment linearised with BgHl, into the BamϊTT polylinker site of pNeo5K-6A. The shuttle vector was used to transform S. lividans TK24 and finally S. clavuligerus WT to thiostrepton (5μg/ml) and neomycin (50μg/ml) resistance. Primary transformants were subjected to two rounds of sporulation under non- selective conditions in order to generate gene replacement mutants as described by Paradkar and Jensen (1995) supra.
4.3 Construction of orfό/orfόpar mutants orfό/ orfόpar double mutants were generated by transforming protoplasts of Hie orfόpar (neor) mutant with the orfό(&nτ ) disruption construct (Mosher et al (1999) supra). Protoplast preparations from orfόpar mutants, were transformed with the orfό disruption construct isolated from S.lividans. Transformants were selected on apramycin (apr) at 50μg/ml. Primary transformants were put through two rounds of sporulation under non- selective conditions in order to generate gene replacement mutants as described by Paradkar and Jensen (1995) supra.
4.4 Fermentation of orfό, orfόpar and orfό/orfόpar mutants
To test the effect of disrupting orfό, orfόpar and orfό/orfόpar on clavulanic acid biosynthesis, spores from each isolate were tested as previously described in section 3.4. Fermentation Analysis of orfό mutants
Mutant 6-1 A defective in the orfό gene was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 57%. From these results it can be concluded that orfό is required for efficient production of clavulanic acid as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation Analysis of orfόpar mutants Mutant 14-2B(2) defective in the orfόpar gene was fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 27%. From these results it can be concluded that, like orfό, orfόpar contributes to clavulanic acid biosynthesis as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation Analysis of orfό/orfόpar mutants
Two separate mutants defective in both orfό and orfόpar were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by an average of 65%.
From these results it can be concluded that both orfό and orfόpar are necessary for efficient production of clavulanic acid since disruption of either copy of the gene causes a reduction in clavulanic acid production. Inactivation of both copies of the gene caused a further decrease, but not a complete loss of clavulanic acid producing ability.
4.5 Southern Analysis The orfό , orfόpar and orfό/orfόpar mutants were further characterised by Southern analysis. The results confirmed that in these mutants the chromosomal copy of the relevant gene had been disrupted as expected.
Example 5 - Analysis of cvmό and cvm6par 5.1 Construction of cvmό mutants
Construction of mutants disrupted in cvmό has already been described in WO98/33896 (cvmό is orfdwn3). 5.2 Construction of cvmόpar mutants
A 1.7 Kb Sail fragment containing cvmόpar was released from pO4H-4 and ligated into pUC118 at the Sail site. The resulting plasmid was digested with iscoNI to release a 140 bp fragment internal to cvmόpar. In place of this fragment, the neomycin resistance gene from pFDNeo-S, released as an EcoKTIPstl fragment, was ligated into cvmόpar after both fragments had been made blunt by treatment with the Klenow fragment of DNA polymerase I. The neoR marker was inserted in tlie same orientation as cvmόpar. The neomycin containing Sail fragment was released with EcoKl and inserted into tlie shuttle vector pUWL-KS (Weimeier, U.F (1995) Gene 165: 149-150.) at the EcόKT site. The construct was named pNeoSall.7U.
The plasmid pNeoSall .7U was used to transform S.lividans TK24, and finally S. clavuligerus wild type. The resulting cvmόpar: :neo transformants were selected on MYM medium with 50μg/ml neomycin and 5μg/ml thiostrepton and then subjected to two rounds of sporulation under non- selective conditions to give double cross-over mutants.
5.3 Construction of cvmό/cvmόpar mutants
The construct pNeoSall .7U isolated from S.lividans TK24 was also used to transform the cvmό mutant 56-3 A, where the apr11 cassette was inserted into cvmό in the same orientation as the gene. Transformants were grown on MYM medium with 50μg/ml neomycin and 5μg/ml thiostrepton. The mutants were put through two rounds of sporulation under non- selective conditions as described above and double cross-over mutants were isolated.
5.4 Fermentation of cvmό. cvmόpar and cvmό/cyrnόpar mutants
To test the effect of disrupting cvmό, cvmόpar and cvmό/cvmόpar on β-lactam biosynthesis, spores from each isolate were tested as previously described in section 3.4. Fermentation Analysis of cvmό mutants
It was reported in WO98/33896 that mutants 56-1 A, 56-3A, 57-1C and 57-2B defective in the cvm6 gene produced elevated levels of clavulanic acid (125-141% of the control strain) and greatly reduced levels of clavam-2-carboxylate and 2- hydroxymethylclavam when cultured in Soy medium.
These results suggest that the cvmό gene is required for efficient production of the 5S clavams. Disruption of cvmό not only results in a reduction in clavams but also a simultaneous increase in clavulanic acid.
Fermentation Analysis of cvmόpar mutants Mutants 3A1, 3A2, 2A-6, 2B-1 and 2B-2 defective in the cvmόpω- gene were fermented in
Soy medium and compared to wild type S. clavuligerus for production of β-lactam metabolites. After 72hrs growth, accumulations in clavulanic acid were increased by 6-11%.
Production of clavam-2-carboxylate and alanyl clavam was abolished and levels of 2- hydroxymethyl clavam reduced by 50-85%.
These results suggest that like cvmό the cvmόpar gene is required for efficient production of the 5S clavams. Disruption of cvmόpar not only results in a reduction in clavams but also a simultaneous increase in clavulanic acid.
Fermentation Analysis of cvmό/cvmόpar double mutants Mutants A-l, A-2, B-l, B-2, C-l and C-2 defective in both the cvmό and cvrnόpar genes were grown in Soy medium and compared to wild type S. clavuligerus for their production of β-lactam metabolites. Production of clavulanic acid was increased by 12-27%, production of alanyl clavam and clavam-2-carboxylate eliminated and levels of 2- hydroxymethyl clavam reduced by 70-83%. These results indicate that, like the cvmό and cvmόpar single mutants, the cvrnόl cvmόpar double mutants produced elevated levels of clavulanic acid and both genes are required for the efficient production of 5S clavams.
5.5 Southern Analysis The cvmό, cvmόpar and cvrnό/ cvmόpar mutants were further characterised by Southern analysis. The results confirmed that in these mutants the chromosomal copies of the relevant genes had been disrupted as expected.
Example 6 - Analysis of orβ and orβpar 6.1 Construction of orβ mutants
Mutants disrupted in orβ were made as described in United States Patent No. 6,332,106.
6.2 Construction of orβpar mutants
The plasmid p5.7£coRI ref (pJOE based hyg) was used as the disruption template for orβpar. The insert in this plasmid is approximately 5.7kb and includes part of cvmόpar, all of orfόpar, orβpar, orβpar and part of orβpar all carried within the plasmid pJOE829 (Kieser, T et al. (2000); Aidoo et al. (1994) Gene. 147:41-6). The disruption vector was constructed by ligation of a thiostrepton resistance cassette (Aidoo et al. supra) into Fsel digested p5.7i?eoRI. A unique Fsel site is located within the insert 507 bp from the start of orβpar. The correct construct was obtained and used to sequentially transform S.lividans TK24 and then S. clavuligerus wild type. Primary transformants were selected on thiostrepton (5μg/ml) and hygromycin (25μg/ml). The mutants were put through two rounds of sporulation under non- selective conditions as described above and putative double cross-over mutants were isolated.
6.3 Construction of orβ/orβpar mutants
The orβpar disruption cassette described in section 6.2 was isolated from S.lividans TK24 and used to transform or 3::apra mutants. Transformants were selected on MYM medium containing thiostrepton (5μg/ml) and hygromycin (25μg/ml). The mutants were put through two rounds of sporulation without selection and double crossover mutants isolated as previously described.
6.4 Fermentation Analysis of orβ, orβpar and orβ/orβpar mutants
To test the effect of disrupting orβ, orβpar and orβ/orβpar on clavulanic acid biosynthesis, spores from each isolate were tested as previously described in section 3.4. Fermentation Analysis of orf 3 mutants
Mutants Ap3-1, Ap3-2 and Ap3-3 were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulations in clavulanic acid were reduced by 31-71%.
From these results it can be concluded that orβ is required for efficient production of clavulanic acid as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation of orβpar mutants
Mutants 3A-1 and 3A-2 were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulations in clavulanic acid were reduced by 9%.
From these results it can be concluded that orβpar is required for efficient production of clavulanic acid as elimination of this gene by disruption causes a reduction in clavulanic acid levels.
Fermentation of orf 3/orβpar mutants Clavulanic acid biosynthesis was completely abolished when mutants 11-1, 11-2, 2-1 and 2-2 defective in both copies of the orβ gene were grown in Soy medium and compared to wild type S. clavuligerus .
These results demonstrate that under the conditions tested, both genes, orβ and orβpar, contribute to clavulanic acid biosynthesis as the double disruption results in a mutant unable to make any clavulanic acid.
6.5 Southern Analysis The orβ, orβpar and orβ/orβpar mutants were further characterised by Southern analysis. The results confirmed that in these mutants the chromosomal copies of the relevant genes had been disrupted as expected.
Example 7 - Analysis of orβ and orβpar
1.1 Construction of orβ mutants
Mutants disrupted in orβ were originally made as described in United States Patent No. 6,332,106. These original orβ mutants were subjected to a second round of gene replacement to remove the apramycin resistance gene and replace it with a simple frameshift mutation. The plasmid construct used to create the original orβ mutant consisted of a 2.1 kb EcoRT/BglR fragment of S. clavuligerus DNA carried on a pUCl 19/pIJ486 shuttle vector, with the orβ gene disrupted by insertion of an apramycin resistance gene cassette into a centrally located Notl site (United States Patent No. 6,332,106). The disruption plasmid construct used in the second round of mutation was derived from the original disruption plasmid by digestion with Notl to release the apramycin resistance gene cassette, treatment with the Klenow fragment of DΝA polymerase I to fill in the overhanging ends, and then re- ligation to circularize the plasmid. The resulting plasmid construct carries the entire orβ gene but with a frameshift introduced at the location of tlie destroyed Ncol site. The construct was used to sequentially transform S.lividans TK24 and then the original S. clavuligerus orβ mutant. Primary transformants were selected on thiostrepton (5μg/ml) and then subjected to two rounds of sporulation under non-selective conditions. Putative double cross-over mutants were identified based on their loss of apramycin resistance .
7.2 Construction of orf2par mutants orβpar mutants were generated using a PCR-based targeting kit known as
REDIRECT (trade Mark of Plant Bioscience Limited, Norwich, U.K). The plasmids pIJ790 and pIJ773, and the host strain E. coli BW25113 were supplied as part of the kit. For this particular application, a pair of oligonucleotide primers, KTA14: 5'-CCATCCCGGCGCCCGTCCGATGCGAAGGAGATCTCCATGATTCCGG- GGATCCGTCGACC-3' and
KTA15 : 5 '-CGGGGCCGGGCATGGTGAACTCGTCCTCCACGGTGGTCATGTAGGC- TGGAGCTGCTT-3', designed to disrupt the orβpar gene by insertion of an apramycin resistance gene, were synthesized. The orβpar disruption cassette was generated by PCR using these two primers with the plasmid pIJ773 as template. PCR conditions used wereas described in the user instructions except that no dimethylsulfoxide was used. The orβpar disruption cassette was then introduced by electrotransformation into E. coli BW25113/pIJ790 which had been previously transformed with the orβpar bearing cosmid 14E10 (described hereinabove). Cosmid DNA was isolated from transformants after overnight growth at 37°C to promote loss of the pIJ790 plasmid and analyzed to confirm that the orβpar gene had been disrupted, orβpar disrupted cosmid DNA was then transferred into wild type S. clavuligerus by conjugation. Conjugation was carried out as described by Kieser, T et al (2000) supra except that AS-1 medium (Baltz, R. H. Genetic recombination by protoplast fusion in Str'eptomyces. Dev. h d. Microbiol 21 (1980) 43-54) supplemented with apramycin at 50 μg/ml was used for recovery of transconjugants. Apramycin resistant S. clavuligerus transconjugants were subjected to one round of sporulation under non-selective conditions in order to generate gene replacement mutants as described by Paradkar and Jensen (1995) supra.
1.3 Construction of orβ/orβpar mutants
The PCR-based targeting procedure used to generate the orf2par mutants (section 7.2) was also used to generate orf2/orf2par double mutants. In this case the orf2par disrupted cosmid DNA was conjugated into the orf2 mutants described above (section 7.1) rather than into the wild type strain. Apramycin resistant S. clavuligerus transconjugants were subjected to one round of sporulation under non-selective conditions in order to obtain unigenomic mutant spores that had undergone gene replacement as previously described.
7.4 Fermentation analysis of orβ, orβpar and orf2/orf2par mutants
To test the effect of disrupting orβ, orβpar and orβ/2par on clavulanic acid biosynthesis, spores from each isolate were tested as previously described in section 3.4. Fermentation Analysis of orβ mutants
Mutants defective in the orβ gene were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulations in clavulanic acid were reduced by 95-98% (Jensen et al (2000) supra.
From these results it can be concluded that orβ is required for efficient production of clavulanic acid as elimination of this gene by disruption causes a severe reduction in clavulanic acid production. Fermentation analysis of orβpar disruptant
Mutants defective in the orβpar gene were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, accumulation of clavulanic acid was reduced by 10-30%.
From these results it can be concluded that, like orβ, orβpar contributes to clavulanic acid biosynthesis as elimination of this gene by disruption causes a reduction in clavulanic acid levels. Fermentation analysis of orβ/orf2par disruptants Mutants defective in both orf2 and orf2par were fermented in Soy medium and compared to wild type S. clavuligerus for production of clavulanic acid. After 72hrs growth, no clavulanic acid production could be detected from the strains contain the or£2 and orf2par mutations. These results demonstrate that under the conditions tested, both genes, orf2 and orf2par, contribute to clavulanic acid biosynthesis as the double disruption results in a mutant unable to make clavulanic acid.
5. Southern Analysis
The orβ, orβpar and orβ/2par mutants were further characterised by Southern analysis. The results confirmed that in these mutants the chromosomal copies of the relevant genes had been disrupted as expected.
Example 8 -Analysis of cvm7 and cvm7par
Sequence analysis had identified two additional genes in the paralogue cluster that did not have obvious paralogues in either the clavulanic acid or cvm gene clusters. It was of interest to determine if either of these genes was a paralogue to an as yet unidentified cvm gene. Therefore the sequence of the cvm cluster (WO98/33896) was extended downstream of cvm3 (orfup3 in WO98/33896).
8.1 Extension of cvm cluster sequence
The cosmid 10D7 (described in WO98/33896) was digested with the restriction endonuclease Sacl. From this digestion a 6.8 kilobase DNA fragment containing casl and cvml was isolated and cloned into a pUCl 19 based plasmid. The resultant plasmid pCEC019 was used as a template to generate sequence information which allowed completion of the partial cvm3 gene reported in WO98/33896. h addition, the sequence information showed the presence of another open reading frame, cvml, which was incomplete in this fragment. In order to complete the cvml gene sequence, the next adjacent Sacl fragment from cosmid 10D7, a 1.9 kb fragment, was subcloned. Sequence information was obtained from the end of the clone which contained the remainder of the cvml gene, up to the point where the start codon for the cvml gene could be identified. In total, this resulted in the generation of a further approximately 3.9 kb of new DNA sequence which is described in Sequence ID No.17.
8.2 Sequence analysis The size of cvml and its orientation relative to the rest of the cvm cluster is showed diagrammatically in fig2. Sequence homology searches demonstrated that this gene shares homology with transcriptional regulator genes. In addition cvml also shared 33% identity with one of the two genes identified in the paralogue cluster that did not have any obvious paralogues within the known clavulanic acid or clavam biosynthetic genes. Therefore since cvmό and cvmόpar have been shown to be paralogues, from this sequence data it can be concluded that cvml and cvml par are paralogues of genes involved in 5S clavam biosynthesis.
Brief description of the figures
Figure 1. Diagram of the paralogue cluster. The orientation of transcription is shown for each gene (direction of arrow) Figure 2. Orientation of cvml in relation to published cvm cluster (WO98/33896). Figure 3. Annotated seqence of the paralogue cluster
Brief description of the sequences
SEQ ID NO: 1 cvmόpara open reading frame SEQ ID NO: 2 cvm7para open reading fame
SEQ ID NO: 3 cvmδpara polypeptide
SEQ ID NO : 4 cvm7para polypeptide
SEQ ID NO: 5 cvm6 open reading frame
SEQ ID NO: 6 cvm7 open reading frame SEQ ID NO:7 cvml open reading frame
SEQ ID NO: 8 cvm2 open reading frame
SEQ ID NO: 9 cvm3 open reading frame
SEQ ID NO: 10 cvm4 open reading frame
SEQ ID NO: 11 cvm5 open reading frame SEQ ID NO: 12 orf2para open reading frame
SEQ ED NO: 13 orf3para open reading frame
SEQ ID NO: 14 orf4para open reading frame
SEQ ID NO: 15 orfόpara open reading frame
SEQ ID NO: 16 paralogue cluster SEQ ID NO: 17 extended cvm cluster (underlined sequence denotes new sequence over that disclosed in WO98/33896
SEQ ID NO: 18 orf2para open reading frame (reverse complement)
SEQ ID NO: 19 orf3para open reading frame (reverse complement)
SEQ ID NO:20 orf4para open reading frame (reverse complement) SEQ ID NO:21 cvm6 polypeptide
SEQ ID NO:22 cvm3 polypeptide
SEQ ID NO:23 orfόpara polypeptide SEQ ID NO 24 orf4para polypeptide SEQ ID NO 25 orβpara polypeptide
SEQ ΓD NO 26 orf2para polypeptide
Sequences
SEQ ID NO: 1 cvmόpara
ATGTTCCACCCGGTCCTGCCCCGGGGCCGCGAGGACCGCACCGTTCTGGTCTCCGGCCGCGGCTGCACCGTACGGGACAC CGAAGGGCGCACCTATCTCGACGCCTCGTCGGTGCTCGGACTGACCCAGATCGGCCATGGACGTGAGGAGATCGCGCAGG CCGCCGCCGAGCAGATGCGGACACTCGGTCACTTCCACACCTGGGGCACCATCAGCAACGACAAGGCCATCCGACTGGCC GCGCGCCTCACCGACCTGGCGCCCCAGGGTCTCCAGCGGGTCTACTTCACCAGCGGCGGCGGCGAGGGCGTCGAGATCGC CCTGCGCATGGCCCGTTACTTCCACCACCGCACCGGCAGCCCGGAGCGCACCTGGATCTTGTCGCGCCGCACCGCCTACC ACGGCATCGGCTACGGCAGCGGTACGGTGTCGGGCTCGCCCGCCTACCAGGACGGGTTCGGCCCGGTGCTGCCCCATGTG CACCACCTCACGCCGCCCGACCCGTACCACGCCGAGCTGTACGACGGCGAGGACGTCACGGAGTACTGCCTGCGCGAACT CGCCCGCACCATCGACGAGATCGGCCCCGGGCGGATCGCCGCGATGATCGGGGAGCCGGTCATGGGCGCGGGCGGCGCCG TCGTCCCGCCGCCGGACTACTGGCCGCGCGTCGCCGCGCTGCTGCGCTCCCACGGCATCCTGCTGATCCTGGACGAGGTC GTCACCGCGTTCGGCCGCACGGGGACCTGGTTCGCGGCCGAGCACTTCGGGGTGACCCCCGATCTGCTGGTGACCGCGAA GGGCATCACCTCCGGGTATGTCCCGCACGGGGCGGTGCTCCTGACCGAGGAGGTCGCGGACGCCGTGAACGGGGAGACGG GGTTCCCGATCGGCTTCACCTATACCGGTCACCCCACGGCGTGCGCCGTCGCGCTCGCCAATCTCGACATCATCGAACGG GAAGGGCTGCTGGAGAACGCGGTGAAGGTGGGCGACCACCTCGCCGGGCGGCTGGCGGCCCTGCGCGGGCTGCCCGCCGT GGGGGACGTCCGGCAACTGGGCATGATGCTCGCCGTCGAGCTGGTGTCGGACAAGACGGCCCGCACCCCGCTGCCGGGCG GCACCCTCGGGGTCGTGGACGCGCTGCGCGAGGACGCGGGCGTCATCGTCCGGGCCACGCCGCGCTCCCTGGTCCTCAAT CCGGCGCTCGTGATGGACCGGGCCACGGCGGACGAGGTGGCGGACGGGCTGGACTCGGTGCTGCGGCGGCTGGCACCCGA CGGGCGGATCGGCGCGGCCCCCCGGCGGGGGTGA
SEQ ID NO:2 cvm7para
GTGTACGAGTGCAGCGATGAGGTTCGTCACGACGTCCCCGGCCTGCCGGGTCCGTCACCGTCCATCACCGTCCTGGGCTG TCTGGGCGTACGCGCCGACGGCCGGAAACTGGAGCTGGGCCCTCCGCGTCAGCGGGCCGTTTTCGCCCTGCTGCTCATCA ACGCGGGCAGTGTGGTGCCGGTCGACTCGATCGTCTTCCGTATCTGGGGCAACTCACCACCGGGCGCGGTCACCGCGACG CTCCAGTCCTATGTGTCCCGGCTGCGGAAACTCCTGGCCGAGTGTGTGCTCCCGGACGGTTCGACACCCGAACTGCTGCA CCAGCCGCCGGGCTACACCCTCGCGCTCGGCACCGAGCACATCGACGCGAACCGTTTTGAGCAGGCCATCAGGACAGGGC GCCGGCTCTCGCGCGAGGAGCAGCACCAGGAGGCGCGGGCCGTGCTCTGCCAGGCCCTGCTGAGCTGGGGCGGGACACCG TACGAGGAGCTGAGCGCGTACGACTTCGCCGTCCAGGAGGCCAATCGGCTGGAGCAGCTCCGGCTGGGCGCCGTGGAGAC ATGGGCGCACTGCTGTCTGCGGCTGGGGCGGGACGAGGAGGTGATGGACCAGCTCAAGCCGGAGGTGCAGCGCAATCCGC TGCGGGAGCGGCTGATCGGGCAGCTCATGCAGGCGCAGTACCGGCTGGGGTGCCAGGCGGACGCGCTCAGGACGTACGAG GCGACGCGGCGGGCCCTGGCCGAGGAGCTGGGGACCGATCCGGGCAAGGAGCTGGCGGCGCTGCACGCGGCGATCCTGCG TCAGGACAACGGTCTGGACCGCGTCGTCCCGGCGTCCGCGCCGCCGTCGGCGGGGGTCGGGCGGGGGGCCGTGACGGTGT CGGTCCCGGCACAGCGGTCGAGGCCGTTGACGCGGCCGGTGGCGGGGCGGGCGCGGGTCCCGGGGGCGATGACGGTGGCG GCGGGCGCGGGGGCGGCCCCCGCGTCCGCCTCCGGCTCCGTTTCCGCGTCCGTTTCCGGCTCCGGCTCCGGCTCCGGCTC CGCTCCTGCGTCGGTTCCCACCTTCTTTCCCGGCTCCGTTTCTGGCTCGGCGTCCGTTGCCGCGTCCGTAGCCGCGCCCG TTTCCGGCCATGTCTCCGGGCCCGGGTCCGCTTTCGGGTCCGTGGCGCTCCACCGGCCGCAGACCCTCCGGGGCGAGCCG GTCCACGGGGGCGCGCAGGGGATGCGCACCGGGCAGGTGTTCCCCACGCTGCCGCCGTTCGTCGGGCGCGGCGACGAGCT GCGCGGTCTGCTGGAGTCCGCGACGTCCGCGTTCCACACCTCGGGGCGGGTGGCGTTCGTCGTCGGCGAGGCGGGCAGCG GCAAGACCCGGCTCCTCTCCGAGTTGGAGCGCTCGGTTCCGGACAGTGTGCGCACCGTCTGGGCGTCCTGTTCGGAGAGT GAGGACCGGCCCGACTACTGGCCGTGGACGACCGTGCTGCGGCATCTGTACGCGATGTGGCCGGAACGTATGCACGGATT CCCCGGTTGGCTGCGGCGGGCACTCGCGGAACTGCTTCCCGAGGTGGGCCCGGAGCCACAGGGGCCGCACTCCCCCGACG GGGGCGAGGAGAACAGCGGCAACGGGGACGGTGCGGGCGACGGGGACAGCACCCCGGCGCACACCCTCACGCTCGCGCCC GCTCTCGCGCCCCCGCGCTCCAGAGAGGCTCGTTTCACCCTGCACGACGCCGTGTGCCAGGCGCTTCTGCGCACGGTCCG CGAACCCGTGGTGATCATGCTGGAGGACATGGAGCGGGCCGACGCCCCCTCGCTCGCCCTGCTGCGCCTCCTGGTGGAGC AACTGCGCACCGTCCCCCTGCTGCTCGTGGTCACCACGCGCACCTTCCGGCTCGCGCACGACGCCGAGCTGCGACGGGCC GCCGCCGTGATCCTCCAGTCGACCGGCGCGCGCCGGGTCCTGCTGAACGCCCTGGACGCACGGGCCACCGGGGAACTCGC CGGAGGGATGCTGGGCAAGGCCCCGGACACCCTCCTCGTACGGGCCCTGCACGAGCGCTCCGCCGGGAACCCGTACTTCC TCGTCCAGCTCCTCCGCTCGCTCCGGCAGGGGCTCGCCGCCGCCTGGGAGACGGAGATCCCGGACGAGCTGGCCGGGGTC GTGCTGCAACGGCTGTCGAGCGTGCCGCCCGCCGTGCGCCGGGTGCTCGACATCTGCGCGGTCGTGGAGCGCAGTTGCGA ACGGCGTGTGATCGAGACCGTGCTGCGCCATGAGGGAATCCCGCTGGAGAACGTCCGTACGGCGGTCCGCGGCGGTCTGC TGGAGGAAGACCCCGACGACCCCGGGCGGCTGAGGTTCGTGCATCCGCTGGTCCGGGAGGCCGTCTGGGACGACCTGGAG AACACCCGTCGGCCCGTSTCVMARGTCCCGTTCCTCCGCGCTCGGGGCGCTGGCCACGGTCTGA
SEQ ED NO: 3 cvmόpara polypeptide MFHPVLPRGREDRTVLVSGRGCTVRDTEGRTYLDASSVLGLTQIGHGREEIAQAAAEQ RTLGHFHTWGTISNDKAIRLA ARLTDLAPQGLQRVYFTSGGGEGVEIALRMARYFHHRTGSPERTWILSRRTAYHGIGYGSGTVSGSPAYQDGFGPVLPHV HHLTPPDPYHAELYDGEDVTEYC RE ARTIDEIGPGRIAAMIGEPVMGAGGAWPPPDYWPRVAALLRSHGILLI DEV VTAFGRTGTWFAAEHFGVTPDL VTAKGITSGYVPHGAVLLTEEVADAVNGETGFPIGFTYTGHPTACAVALANLDIIER EGLLENAVKVGDHLAGR AALRGLPAVGDVRQ GMMLAVELVSDKTARTPLPGGTLGVVDALREDAGVIVRATPRS VLN PA VMDRATADEVADGLDSVLRR APDGRIGAAPRRG
SEQ ED NO:4 cvm7para polypeptide
VYECSDEVRHDVPG PGPSPSITVLGCLGVRADGRK ELGPPRQRAVFAL LINAGSVVPVDSIVFRIWGNSPPGAVTAT
LQSYVSRLRKLLAECVLPDGSTPEL HQPPGYTLA GTEHIDANRFEQAIRTGRR SREEQHQEARAV CQA SWGGTP YEELSAYDFAVQEANRLEQLRLGAVET AHCC RLGRDEEVMDQLKPEVQRNPLRER IGQLMQAQYRLGCQADALRTYE
ATRRALAEELGTDPGKELAALHAAILRQDNGLDRWPASAPPSAGVGRGAVTVSVPAQRSRPLTRPVAGRARVPGAMTVA AGAGAAPASASGSVSASVSGSGSGSGSAPASVPTFFPGSVSGSASVAASVAAPVSGHVSGPGSAFGSVALHRPQT RGEP VHGGAQGMRTGQVFPTLPPFVGRGDE RGLLESATSAFHTSGRVAFVVGEAGSGKTRLLSELERSVPDSVRTVWASCSES EDRPDY P TTVLRHLYAM PERMHGFPGWLRRALAEL PEVGPEPQGPHSPDGGEENSGNGDGAGDGDSTPAHTLTLAP ALAPPRSREARFTLHDAVCQALLRTVREPVVIMLEDMERADAPSLAL RLLVEQ RTVPLLLWTTRTFRLAHDAELRRA AAVILQSTGARRV LNALDARATGELAGGMLGKAPDTL VRA HERSAGNPYFLVQLLRSLRQG AAAWETEIPDELAGV VLQRLSSVPPAVRRV DICAVVERSCERRVIETVLRHEGIPLENVRTAVRGGLLEEDPDDPGRLRFVHPLVREAV DD E NTRRPVSRSSALGA ATV
SEQ ID NO:5 cvmό GTGCCCGGCTCCGGACTCGAAGCACTGGACCGTGCCACCCTCATCCACCCCACCCTCTCCGGAAACACCGCGGAACGGAT CGTGCTGACCTCGGGGTCCGGCAGCCGGGTCCGCGACACCGACGGCCGGGAGTACCTGGACGCGAGCGCCGTCCTCGGGG TGACCCAGGTGGGCCACGGCCGGGCCGAGCTGGCCCGGGTCGCGGCCGAGCAGATGGCCCGGCTGGAGTACTTCCACACC TGGGGGACGATCAGCAACGACCGGGCGGTGGAGCTGGCGGCACGGCTGGTGGGGCTGAGCCCGGAGCCGCTGACCCGCGT CTACTTCACCAGCGGCGGGGCCGAGGGCAACGAGATCGCCCTGCGGATGGCCCGGCTCTACCACCACCGGCGCGGGGAGT CCGCCCGTACCTGGATACTCTCCCGCCGGTCGGCCTACCACGGCGTCGGATACGGCAGCGGCGGCGTCACCGGCTTCCCC GCCTACCACCAGGGCTTCGGCCCCTCCCTCCCGGACGTCGACTTCCTGACCCCGCCGCAGCCCTACCGCCGGGAGCTGTT CGCCGGTTCCGACGTCACCGACTTCTGCCTCGCCGAACTGCGCGAGACCATCGACCGGATCGGCCCGGAGCGGATCGCGG CGATGATCGGCGAGCCGATCATGGGCGCGGTCGGCGCCGCGGCCCCGCCCGCCGACTACTGGCCCCGGGTCGCCGAGCTG CTGCACTCCTACGGCATCCTGCTGATCTCCGACGAGGTGATCACGGGGTACGGGCGCACCGGGCACTGGTTCGCCGCCGA CCACTTCGGCGTGGTCCCGGACATCATGGTCACCGCCAAGGGCATTCACCTCGGGGTATGTGCCGCACGGCGCCGTCCTG ACCACCGAGGCCGTCGCCGACGAGGTCGTCGGCGACCAGGGCTTCCCGGCGGGCTTCACCTACAGCGGCCATGCCACGGC CTGCGCGGTGGCCCTGGCCAACCTGGACATCATCGAGCGCGAGAATCTGCTCGACAACGCCAGCACCGTCGGCGCCTACC TGGGCAAACGCCTGGCCGAGCTGAGCGATCTGCCGATCGTCGGGGACGTCCGGCAGACCGGTCTGATGCTCGGTGTCGAA CTGGTCGCCGACCGCGGAACCCGGGAGCCGCTGCCGGGCGCCGCCGTCGCCGAGGCCCTGCGCGAGCGGGCGGGCATCCT GCTGCGCGCCAACGGCAACGCCCTCATCGTCAACCCCCCGCTGATCTTCACCCAGGAAGACGCCGACGAACTCGTGGCGG GCCTGCGCTCCGTACTCGCCCGCACCAGGCCGGACGGCCGGGTGCTCTGA
SEQ ID NO:6 cvm7
ATGAAGTACGACATAACCCCACCATCCGGCCTTCGGTTCGACCTCCTCGGCCCGTTGACCGTGACCGCCGGCGAGCAACC CGTGGACCTGGGCGCGCCACGGCAGCGCGCCCTGCTCGCCCTGCTGCTCATCGATGTCGGCAACGTGGTCCCGCTGCCGG TCATGACCGCGTCGATCTGGGGGGCCGACCCACCGTCCCGGGTCCGGGGGACGCTCCAGGCTTATGTGTCCCGACTGCGG AAACTCCTGCACCGCCATGACCGTTCCCTTCGCCTTGTCCACCAGCTCCAGGGGTATCTCCTCGAAGTGGATTCGGCGAA GGTGGACGCCGTGGTTTTCGAGACACGTGTCAGGGAGTGCCGGGAATTGAGCAGGGCCCGGAACCCCGAGGCCACCCGGG CCGTGGCCTGGTCCGCCCTGGAGATGTGGAAGGGCACACCCATGGGCGAGCTGCATGATTATGAATTTGTGGCGGCGGAG GCCGACCGGCTGGAAGGAATCCGGTTACGCGCGCTGGAGACCTGGTCCCAGGCGTGTCTCGATCTCCAGCACTATGAAGA GGTTGCATTTCAGCTCGGCGAGGAGATCCACCGCAATCCGGAACTGGAACGGCTGGGCGGTCTCTTCATGCGGGCCCAGT ATCATTCCGGACGGTCGGCGGAAGCCCTGTTGACGTATGAACGTATGCGTACCGCGGTGGCGGAGAATCTGGGGGCCGAT ATCAGTCCGGAGCTCCAGGAACTCCATGGAAAGATTCTGCGCCAGGAACTCACGGAGACACCCGCCGCGCGATCGACGGC CTCCCTCACACGGGCGGCGGGCCCGCACGGGCCCCCGCCCCTGGCCGAAACCGGCACCCCCGCCGCACCCGCGGACATGG CCGAAACCACGGTGGCGGAGGAAAGCGCCGCGCCCCCCGCCCCGGCGGCGCCCGGGACCCCGCCCCCCATGCCGTCCCCC GTACCGCTCCCCCATCCGTCAGGGGCCGTCCCGCCGGTCACCCCGGTGCCTCCCCCGGTCCCCCGCTCGGCCCTCCGTTC AGCGGCACCCGCCGAGACCGAGGACCCGGAACCGGCGCCGCCCCCTCCCCCTCCGCCGGGCGGCCGACTCATCGGCCGCC GCGCCGAACTGCGCAGGCTGCGGCTGCTGCTGACGAAGACCCGCGCGGGCCACGGCCATGTCCTGCTGGTCTGCGGCGAA CAGGGCATCGGGAAGACCCGGCTCCTGGAGCACACCGAGCACACCCTGGCCGCGGGCGCGTTCCGGGTGGTCCGTTCGCA CTGCGTCGCCACCCTCCCGGCACCGGGCTACTGGCCCTGGGAGCACCTCGTACGCCAGCTCGACCCGGACAGCGGCCTCG GTGACGACGGCGACGCCGACCCCGTCGCCCAGGCCGAGTGGCTGCCGGAACACCACCTCACCCACCAGATGCGGATCTGC CGGACGGTGCTCGCCGCGGCGCGGCGGACCCCGCTCCTGTTGATCCTGGAGGATCTGCACCTCGCCCACGCGCCGGTCCT GGATGTGCTCCAGCTCCTGGTCAAACAGATCGGCCAGGCCCCCGTCATGGTCGTCGCCACCCTGCGCGAGCACGATCTCG CCCGGGACCCCGCCGTCCGCCGGGCCGTGGGCCGCATCCTCCAGGCGGGCAACACCGGCACCCTCCGGCTGGACGGGCTC ACCGAGGAGCAGAGCCGGGAGCTGATCGTCTCGGTCGCGGGGGCCCCGTTCGCGCCCCATGACGCCCAACGGCTCCAGCG CGCCTCGGGCGGCAACCCGTTTCTGCTGCTCAGCATGGTCACAGGGGAGGACGGCACCCAGGAGTGGGCACGGCCGTGCG TCCCGTTCGAGGTGCGCGAGGTGCTGCACGAGCGGCTGAGCGAATGCTCCCCGTCCACCCAGGACGTGCTCACGCTCTGC GCCGTGCTCGGCATGAGCGTGCGCCGACCGCTGCTCACCGACATCATGTCCACGCTCGACATCCCGCACACCGCGCTCGA CGACGCGCTCGGCACGGGGCTGCTGCGCCACGACCGGAACACCGACGGAATGGTCCACTTCGCCCATGGGCTGACCCGGG ACTTCCTGCTCGACGACACCCCGCCGGTCACCCGCGCCCGCTGGCACCACCGGGTCGCCGCCACCCTCGCCCTGCGCTTC CAGCAGGGCGACGACCACGCCGAGATCCGCCGCCACTGTCTGGCCGCGGCCCGTCTGCTCGGCGCCCGCGCGGGGGTGCG CCCCCTGCTGGCGCTGGCCGACCGGGAGCAGTCCCGCTTCTCCCACGCGGAGGCGCTGCGCTGGCTGGAGAGCGCGGTCG CGGTCGTCGCGGCGCTGCCCCGGGACCAGCCGGTGTCCGCCGTCGAACTCCAGTTGCGCAAACGGATGATGGCGCTGCAC GCGCTGATGGACGGCTATGGATCGGCCCGCGTCGAGACGTTCCTCTCCCAGGTCACCCAGTGGGAACACGTCTTCGACAA CACCCAGCCCACCGGGCTGCTGCACGTCCAGGCGCTGAGCGCGCTCACCACGGGCCGCCATGAGCAGGCGGCGGAGCTGG CCGGGCTGCTGCACGAGCTGGCCGACCACGGCGGCGGACCGGAGGCCCGGTCGGCGGCCTGCTATGTGGACGGCGTCACC CTGTATGTGGGCGGACGGGTCGACGAAGCCCTCGCCGCGCTCGCCCAGGGCACCGAGATCACGGACGCCCTCCTGGCCGG ACACCGCAGGACCGCCGCCCCGCACGGCGGCGGGCACCTCCAGGACCGGCGTATCGACTTCCGCGCCTATCTGGCGCTCG GCCACTGTCTCAGCGGCGACCGGATTCAGACCCAGCGCTACCGGACGGAACTCCTCCACCTCACCCAGTCGGAACGGTAC GACCGGCCGTGGGACCGGGCCTTCGCCCGCTATGTGGACGCGCTCATCGCCGTCACGGAGTGCGATGTCCAGGGGGTGTG GCTGGCCGCGCGGGCGGGGCTCGACCTCGCCGCCCGCTGCCAGCTCCCGTTCTGGCAGCGGATGCTCGCCGTCCCCCTCG GCTGGGCCGAGGTCCACCAGGGGGCGCACGACAAGGGGCTGGCCCGGATGCGGGAGGCGCTGCACGAGGCGGCCCGGCAC CGGACCCTGCTGCGCCGTACGCTCCACCTCGGCCTGCTCGCCGACGCCCTCCAGTACACGGGCGCCCGGGAACAGGCCCG GCGCACGATGTCCTCCGCCGTACGGGAGATCGAGCGCCGCGGCGAGTACTTCTGTCTCCGGCCGCAGTGGCCCTGGGCCC GGCTCCTCCACAGCCACGGCACCTCCGCCGCGGCGGAGCACCGGGTCGTCCACGGCAGGCACTGA SEQ ID NO:7 cvml
ATGTCCCGCTCTCCGCCCGAGTCCCCGGCCGGTTCCGTGTCCGCCGCGGTTCCGCGTCCGCCGGTCCGCGCCCTGCGGGA CCTTCCGGTCAGTGCCCAGGGGCTCGGCTGCCTGCCGACCACCGACTTCTACGGACGCCCGGACCGCGCCCGGGCGACGG CCACCATCCGCGCCGCCGTCGACGCCGGGGTCACCCTGCTGGACACCGCCGACGTCCAGGGGCTCGGCGCCGGTGAGGAG CTGCTCGGACGGGCGGTCGCGGGCCGCCGGGACGAGGTGCTGATCGCCACCAAGTTCGGCATGGTGCGCTCGTCCGACGG CGCCTCCCAGGGCTTGTGCGGCGAGCCGTCCTACGTCCGCGCGGCCTGCGAACGGTCCCTGCGTCGTCTCGGCACCGACC GCATCGACCTGTACTACCAGCACTGGACGGACCCGGCGGTGCCGATCGAGGAGACCGTGGGTGCGGTGGCCGAGCTGGTG CGCGAGGGCAAGGTCCGCAGGCTCGGTCTCTCCGAGCCCTCCGCGGCCACGCTGCGCCGGGCGGACGCGGTGCACCCGGT GACGGCGGTGCAGAGCGAGTGGAGCCTGTGGTCGCGCGGGATCGAGGACGAGGTGGTGCCCGTCTGCCGGGAGCTGGGGA TCGGGATCGTCGCTTACGCCCCTCTGGGACGGGGTTTTCTCACCGGCACCATCCGCACCACCGACGATCTGGGGGACGAG GACTTCCGCCGGGGCCAGCCCCGGTTCAGCGCTCCGGCCCTCGCGCGCAACCGCTCGTTGCTGCACCGGCTGCGCCCGGT CGCGGACGGTCTGGGGCTGACCCTGGCACAGCTCGCGCTCGCCTGGCTGCACCACCGGGGCGAGGACGTCGTCCCGATCC CGGGCACCGCGAACCCGGCCCATCTCGCGGACAATCTCGCCGCCGCCTCGATCCGGCTGGACGACCGGTCCCTCGCGGAG GTGACGGCCGCGATCTCCCACCCGGTGTCCGGGGAGCGGTACACCCCGGCATTGCTCGCCATGATCGGCAACTGA SEQ ID NO:8 cvm2
ATGTCCGTGGCATCGGCCGGTATGACGGACGAGCAGCGCAAGGCGGTCATCACCGCGTACTTCAAGGCGTTCGACAACGG CGGCGTCGGCAGCGACGGCACCCCCGCGATCGACTACTTCGCCGAGGACGCGGTCTTCTTCTTCCCCAAGTGGGGTCTGG CCCGGGGCAAGTCCGAGATCGCCCGGCTCTTCGACGACCTCGGGGGCACCATCCGCTCGATCACCCACCATCTGTGGTCC GTCAACTGGATTCTGACCGGGACCGAACTCCTCGCCGCGGAGGGCACCACCCACGGTGAGCACCGGGACGGGCCGTGGCG GGCGGGTGACCCCGAGTGGGCCGCCGGGCGCTGGTGCACGGTCTACGAGGTGCGGGACTTCCTCGTCCACCGGGCCTTCG TCTATCTGGACCCCGATTACGCGGGCAAGGACACCGCGCGTTACCCGTGGCTGTGA
SEQ ID NO:9 cvm3
GTGACCCGGCCTCCGGGCCTTTCCGCGCACACCCACGGGTCCGTGTCCGGGAGTCTGCTGCGCCGGGTGGCGGGCCACTA TCCCACCGGGGTGGTCCTGGTCACCGGTCCGGCCGAGGCTCCGGGGCAGCCGCCGCCCGCCATGGTGGTGGGGACGTTCA CCTCGGTGTCGCTCGATCCGGTGCTGGTGGGTTTCCTCCCGGCCAGGTCGTCGACGACCTGGCCGCGGCTCCGGGCGGCC GGGCGTTTCTGCGTCAATGTGCTCGGCGCGGATCAGGGCCCGGTCTGCCGGAGTTTCGCCGGGGGCGATCCGGGGCGCTG GGAGGTGCCGTACCGGACGACGGCCACCGGCTCCCCCGTCCTGCTCGACGCGCTCGCGTGGTTCGACTGCGAGGTGGCGG GGGAGACGGAGGCGGGCGACCACTGGTTCGTCACCGGGGCGGTGCGCGACCTCGGGGTGATCCGCGAGGGTTCGCCCCTG GTCTTCCTGCGGGGCGACTACGGGCACTGGGCCGGGGGCGGCGGCTCGGGCCGGGCGGGGCGGCGGTCCGCCGTCTGCCC GGTCTGA
SEQ ID NO: 10 cvm4
GTGGAATGCCGCATATTCGAGATCGACGAACTGCCGTTGCTGGACGGGGAGGTCCTGCGGGACGCCCGGATCGGTTACGC CATGTACGGCACGCCGAACGCCGACGGGACGAACGTGGTGCTCTGTCCGTCGTTCTTCGGCCGGGACCACACCGGGTACG
ACTGGCTGATCGGTGCGGGGCTGCCGCTGGACACCCGGCGGTACTGCGTCGTCACCGCCGGACTCTTCGGCAACGGGGTC
TCCAGCTCGCCCGGCAACCACCCGTCGGGGTCCCGCTTTCCGCTGATCACTCCGCAGGACAATGTCGCGGCGCAGCACCG
GCTGCTCACCGAGGAGCTGGGGGTACGGGAACTGGCCCTGGTCACGGGCTGGTCGATGGGCGCGGCCCACGCCTACCAGT
GGGCCGTGTCGCATCCGGGGATGGTGCGCCGGATCGCCCCGATCTGCGGGGCGCCGGTGAGCAGCCCGCACAGCCTGGTC CTGCTGTCCGGTCTGGCCGCGGCGCTCAGCGCCGACGCCGGGGAGCGGGGGCGGAAGGCGGCGGGCCGGGTGTTCGCCGG
GTGGGGGACCTCGCGTTCCTTCTGGGCCCGCCGTGCCCACCGGGAGCTGGGTTTCGCCACCCGCGAGGAGTACCTCACCG
GCTTCTGGGAGCAGGTCTTCCTCTCCGGGCCCGGCGCCGCGGATCTGCTCACCATGGTGCGCACCTGGGAGAACACGGAT
GTGGGGGCGACACCCGGGGCCGGGGGGAGCGTCGAGGCGGCGCTGGCCTCCGTCACGGCGCGGGCCGTGGTGCTGCCGGG
CGCCCTGGACGTGTGTTTCGCCGTCGAGGACGAGAAGCGGGTGGCCGATCTGCTGCCGTATGCCTCGCTGGAGGTGATCC CGGGAGTGTGGGGGCATCTCGCGGGGTCCGGGGGGTCGGCCGCCGACCGGGAGTTCATCGGGGGCGCGCTGCGGCGGCTG
CTGGACAGCCCGGTGGACGGGGGCTGA
SEQ ID NO: l l cvm5
GTGAAGTCCATTCTCTTCTATCTGCCAACGGTCGGCAGTCATGCGCAGGTCCAGCGGGGTATGGCGGGGGTCAATCCGCA GAACTACCAGAACATGCTCCGGCAGCTCACCCGGCAGGCGCAGGCGGCCGACGAACTCGGCTACTGGGGACTGTCCTTCA
CCGAGCACCACTTCCACACCGAGGGTTTCGAGGTCTCCAACAACCCGATCATGCTGGGGCTCTACCTCGGCATGCAGACC
CGGCACATCCGGGTCGGCCAGATGGCCAACGTCCTGCCGCTGCACAATCCGCTGCGGCTGGCCGAGGATCTGGCGATGCT
CGACCACATGACCCGGGGCCGCGCCTTCGTCGGGATCGCGCGCGGGTTCCAGAAGCGCTGGGCCGACATCATGGGGCAGG
TGTACGGGGTCGGCGGCACCCTGTCCGACGCCGGGGAGCGGGACCGGCGCAATCGTGCCCTCTTCGAGGAGCACTGGGAG ATCATCAAGAAGGCGTGGACGACCGAGACGTTCACCCACTCCGGGGAGCAGTGGACGATCCCGGTGCCGGACCTGGAGTT
CCCCTACGAGGCGGTGCGCCGCTACGGCCGGGGCCTCGACGAGAACGGCGTCATCCGCGAGGTGGGCATCGCGCCCAAGC
CCTACCAGCGCCCCCACCCGCCCGTCTTCCAGCCGTTCAGCTTCAGTGAGGACACGTTCCGGTTCTGTGCCCGGGAGGGC
GTGGTGCCGATCCTGATGAACACCGACGACCAGATCGTCGCCCGGCTGATGGACATCTACCGGGAGGAGGCCGAGGCGGC
GGGCCACGGCACCCTGCGGCGGGGCGAGCGGGTCGGGGTGATGAAGGACGTCCTGGTCTCCCGGGACTCCGGCGAGGCCC ACCACTGGGCGTCCCGCGGCGGCGGCTTCATCTTCGAGAACTGGTTCGGCCCCATGGGCTTCACCGAGGCGCTGCGCGCG
ACCGGCGAGACGGGTCCGATCGGCTCGGACTACAAGACCCTGGTCGACCGGGGGCTGGAGTGGGTCGGCACCCCGGACGA CATCAACCGCATGATCGAGAAGCTGGTGGAGCGGCACGATCCGGAGTATCTGCTCCAGTGCCAGTACTCCGGGCTGATCC CGCACGATGTCCAGCTGCGCAGCCTGGAGCTGTGGGCCACCGAGATCGCCCCCAACTGGCTCTGA
SEQ JJD NO: 12 orf2para TCAGATGGCCAGGGCGGCGAAACCGCCGGACTGGAAGTCGTAGGCCACCGGTACCTCGATCAGGAACGGGCGGCCGAGTC CGGCGCCCTTGGTGAGGGCGGCGAGCAGCGAGGTGCGGTCGGTGGCGCGGACGGCCTCGCAGCCGTTGGCCTCGGCGAGC TGGACGAAGTCGACGCTTCCGAAGCCGACGGCGGGGGCGTGGGAGCGCTGGTGTCCGAGGTTCTGGTACAGCTCGATCAG GCCGTTGCGGTCGTTGTTGACGACGACCATGACGATCGGCAGGCCCAGGCGCACGGCCGTCTCGATGTCGGCGCTGTTGG AGTGGAAGCCGCCGTCGCCCGCGATGAGGAAGACGGGCTCGCCGGGCCGGGCGATCTGGGCGGCCATGGCGGCGGGCAGT CCGTAGCCGAAGCTGGAGCAGCCCGCGGAGGTGAGGAATCCGTACGGCTGGTCGGACTTGGCGAAGAGCACGCCGTAGTG GCGGAAGAAGCCGATGTCGCTGACGAAGGTGCCGTTGTCGAGGACGGAGTTCATGCAGTCGATCACCTGGTGGACCCGCA TGCCGTCCTCGTACTCGGTGGGGTCGGCGAGGAATTCGGCGACGCGGGCGCGCAGGGCGCTGAGGTCGTGCCGGGTCTTG GGGGCGAGGCCCGAGGTCGCGTCGTCGAGCGCGGTGACGAATTCGGCGACGTTGGTGACGATGTCGATGTCGGCGCGGAA CAGCTCCGGGATCGGGTTGACCTCGGGGGCGACCCGGACCGTGGTCTTGGCCCGGCCCCGCGTCCACATGGAGGGGCGCA GGTCCTCGGCGTAGTCGTAGCCGATCGCCAGGAGGAGGTCGGCGGGGCCGAAGATCTCGTCGAGGGCCGGGTGGCCGAGA ATGCCGTCCATGTAGCCGCTGATGGCGCCGTAGTTGAGCGGGTGGTCGTGCGGCAGGACGCCCTTGGCGGTGTAGGTGGT GACGACGGGGATGTTCAGCCGCTCGGCGAGGGCGCGCAGGGCGTCGACGGCCCCGGCGCGGATGACGGCGCTACCGACGA CGAGGAGGGGGTTCTCGGCCTCGCGCACCAGCTCAGCGGCCTCGTCGAGGCGGGCGCGCCAGTCGGCGTCCAGGGCGTGG GTGGCGGTGGCCCGGACCAGGGGGGCGTCGGTGGGGGTGCCGTTCAGCTCGGCGCCGAGGAGGTCGACCGGCAGGCTGAT GAAGCTGGGACCCACGGGCTCGATCCGGCTGTTGAGGACGGCGCTGTCGACGAGGTTGACGATGTCCTCGCCGCGTTCGA GCTGGACGCTGAACTTGGTCAGCGGGCCCATCACGGCGGTGCTGTCCAGGCACTGGTGGGTGACGTTGGGGTAGCAGTCG TACGACTCGGACTGCGCGGCCAGCGCGATGACCGAGCTGCGGTCCAGGGCGGAGGTGGCGACGCCGGTGGCCAGGTTGGT CATGCCGGGGCCCAGGGTCGCGAAGCACGCCTGGGGGCGGTTGGTGATCCGGGCGAGGACGTCCGCCATCACCCCGGCGG TGAACTCGTGCCGGGTCAGGACGAAGTCGAGTCCTTCGACCTCGTCGAAGAGAATGGCGGACGCCTCCCGGCCGACGACG CCGAATACATGGTCGACACCGTACTGGTGAAGACGTTCCAGCATGGCTTTCGCGGTCGTGGTGGCCAT
SEQ ID NO: 13 orβpara
TCATACGACCACCCGGCCCTGGAGCCTGAGCCTGCGCACCGCGTCGACGGAGCGCCGCACCGTCTCGCCGAAGTCCACGT CCTCCGGCGGCACCGTGTCGATGACCACCGCGTCGTACAGGCGCCGTGCCATGGCGCCCTTGACGGCCGTCACCTCGTCG CGCCGGATCCCTTCGGCGAGGAGCAGTCCGGTCCACGCGCTGGTGGTGCCGGACCCCTCGTGGATGCCCAGCTTGGGGCG GGCCACGGTCTCGGCGGGCAGCAGGCCGGAGAGGGCCTGCCGCAACACCCACTTGTCGGTGCCCCGCCGGCGTTTGAGCC CGGGTTCGAGGGAGACCAGCGCGTCCAGGACCGCGCGGTCCCAGTACGGGTGGGTGGTCCACTTCCCGGCGATGCCCGCG AGGACGGGGGACATCTCGTTGAGGCCGTCGAAGCCCGCCATGTCGCCCGCGATCTCGTCGTCGAGGGACCAGAGCGAGGC CGTGCGCCGGTGCATACCGCCGAGCGGGATGTCGGCGCCGTACCCGGTGAGGATGCGGAGCGGCCCGGTGTCGAGCCGCC GGTAGAGGGCGACGAGCGGCAGCAGGTACTCCAGGACCGTGGGGTCGGTGATCTCCGCGGCGGCGACCGCCCAGGGCAGT TCCCTGACGAGTTCGGCCGAGTGGAGCCGGATCTCGCTGTGCGCGGTGCCCAGGTGGACGGCGACCGAGCGGGCCGCGTC GAACTCGTCGGACACCTCGGTGCCCATCGACACGGACCGTGTCCCGGGTGCCAGGGCCGCCGTGTGGGCGGCGACTCCCC CGGAGTCGATGCCGCCGGACAGGACGACGGTGGGGGCCGCCTCCCCGCCGCGCAGCCGGGTGCGGACCGCCGTGGCGAGG CGTTCGCCGACCAGGTCCACCGCCTCCCGTTCGCCGGGCAGCGCCCGGGAGAGCGGGGGTGTCCAGGTGCGGACCGCCCT GGCGGTGATGTCGGAGCCGCCGACTCCGTGCAGCAGGAGGGCGGTCCCGGCGGGGACCCGGCAGACGCCCGCCGCCCCCG GCGCGGTGTGGGTGCCGGACAGGCCCAGCGGCCGGCCCGGCTCGTGCGCCAGGGTCTTCGCCTCGGTGGCGGCGCTCAGC CCCGTCACGTCGGCGCGCAGCCACAGCGGTACCGAACCGGCGTGGTCGGTGGCCGCGACGGTCGCGCCGGTGGAGGCGTC GGTGAGCAGTGCGGCGAACCGTCCGTTCAGGAGCCGGAAGGCCCCGGGGCCCCAGCGCCGCCAGGCGGCCAGCAGCAGTT CGGCGTCGCCGAGGGCGGCAGAGGAGCCGCCGAGCGCTCCGGTCAGCTCGGCGCGGTTGTACAGCTCGCCCGCCAGGAGC AGCCGGACCTGGCCGTCGGCGACCAGGACGGGCGGACGGCCCAGGGTCACGGCCGTTCCGCTCCAGAGCGGGTACGCGGT GCCGTCGTGCACGGGGACATGGGTCCCGCGGACGGCGAAGCGGGGTGCGCTGCCGGGTTCGGAGTGACCGCCGGGGCCGC CGCCGGGGCGGCCCTCGGTGCCGATGCGCACCCGGAATCCGTACACGAGGTCGGGGCCGGGCAT
SEQ JD O:14 orf4para CTACCCCCACCGCTGCCCGGCGAAGTCCACGGCGCTCTCGGCGTCCACCGCGTCCACCGCGTTCTCGGCGTTCTCGGCGT CGTCCGCCGCCGCCCCCGGTGGCAGGGGAGAGTCCACCGGTGCCGACGCGGGCGACGTGGTGGCGCGGGCGTACTGGTAG AGCAGTTCGGCCCCGATCTCCGCCGCCAGCAGGGAGGTGATCCCCGACGGGTCGTACGCCGGGGACACCTCGACCACGTC GAAGCCGACGGGCCTGAGCTGCCCGACCACGTCGAGCAGGGTCAGCACCTCGCGCGAGGACAGCCCGCCGGGGGCCGGTG TGCCGGTGCCCGGGGCGTACGCCGGGTCGACGACGTCGATGTCGACGGAGACGTACAGCGGCAGGCCGCCGACGGTGCGC CGGATCTGCTCGGCGATGCCGCGCGGTGAGCGCCGGGTGAAGTCGGCGGCGGTGACGATGCTGACGCCGTGCCCGCGCGC GTAGTCCAGGGAGTCGGGCCGCGGATTGTGGCCGCGGATGCCGACCTGGACCAGGCGCTCCGGGTCCACCAGGCCCTCTT CGATGGCCCAGCGGAAGGGGGTGCCGTGGTGGTAGGTGCCGCCGTAGACGGGTGGGTTGGTGTCGCTGTGCGCGTCCAGG TGCAGGACGGCGACCCGGCCGTGGCGGGCGTGCACGGCGCGCAGGGCGGCCAGGGAGAGCGAGTGGTCCCCGCCCAGCAT CAGGAACGCGTCGTTGCGTTCCAGGAGCCGGGTCAGGGCGACCGTCGCGGTGTCCATCGCCAGGTCCATCGAGAAGGGGC TGAGGTCGATGTCGCCCCCGTCGACCACGTCGATCCGGTCGAAGACCCCTGGGCCCCGGTCGATGCCGACGCCGTGGATC AGGCTGGACTCGTGCCGGATGGCGCGCGGCGCGAACCGCGCGCCGGGCCGGTAGCTGGTGCCTCCGTCGTACGGGGCGCC GACGACCACCACGTCATGGCCGATCGGGTCGGGCCGGTGGCGCAGCCGCATGAAGGTCGCCGGTTGGGCGTAGCGCGGGG AGACGGCGGTGGACAC SEQ ID NO: 15 orfόpara
ATGCGTGCCTCTTCGCCCAGAGGGTTCCGCGTGCACCACGGTCACGCCGGGATCAGGGGGTCCCACGCGGACCTCGCCGT CATCGCCTCCGACGTTCCCGGGGCGGTCGGCGCGGTGTTCAGCCGTTCGCGGTTCGCCGCGCCGAGTGTGCTGCTCAGCC GGGACGCGGTCGCCGACGGGATCGCCCGGGGCGTGGTGGTGCTGTCCGGCAACGCCAACGCCGGGACGGGCCCGCGGGGG TACGAGGACGCCGCGGAGGTGCGCCATCTGGTGGCCGGGATCGTCGACTGCGACGAGAGGGATGTGCTGATCGCCTCCAC GGGACCCGTCGGCGAGCGGTATCCGATGTCCCGTGTCCGGGCCCATCTGCGGGCGGTGCGCGGGCCCTTACCGGGTGCCG ACTTCGACGGCGCGGCGGCGGCCGTGCTGGGCACCGCGGGCGCCCGTCCCACGATCCGGCGGGCGCGGTGCGGCGACGCG ACGCTGATCGGTGTCGCCAAGGGCCCGGGTACGGGCCCGGCGGAGCAGGACGACCGGTCGACGCTGGCGTTCTTCTGCAC GGACGCCCAGGTGAGCCCCGTCGTCCTCGACGACATCTTCCGCCGGGTCGCGGACCGCGCCTTCCACGGGCTGGGCTTCG GCGCCGACGCCTCCACCGGCGACACGGCGGCCGTTCTCGCCAACGGGCTCGCGGGCCGGGTGGACCTCGTCGCGTTCGAA CAGGTCCTGGGCGCGCTGGCGCTGGACCTGGTCAGGGACGTCGTCCGGGACAGCGGCTGCGGCGGCGCCCTGGTCACGGT GCGGGTCACCGGGGCCCACGACACCGAGCAGGCCGGGCGCGTGGGCCGGGCGGTGGTCGACGCGCCGTCGCTGAGGGCCG CGGTGCACGGCCCGGCACCCGACTGGGCGCCGGTCGCCGCCGTGGCGGGTGGACACGGGGACGAAGGCCCCGGCCGGTCT CCCGGGCGGATCACGATCCGGGTCGGCGGCCGGGAGGTCTTCCCCGCCCCCCGCGACCGGGCCCGCCCGGACGCCGTCAC CGCGTATCCGCACGGCGGCGAGGTGACCGTCCATATCGACCTCGGTGTCCCGGGCCGGGCGCCCGGCGCGTTCACGGTCC ACGGCTGCGACCTCCTGGCGGGGTACCCGCGCCTCGGCGCCGGCCGGGCCGTCTGA SEQ ID NO : 16 para cluster
CCATGGGAGCAGCATCGCAGTGCGCCTCCCCGGCCGCCATGCCGCTAGCTGGTAGTCCCCCTGCCGGGTGCCGACCGCCG GGGCGGTCCCGGGTGCGGCGGCCGGATCTAGTCGGTGTGCTCCGACGGTGCCTGCTGGGTGAGGGGCAGTGTCAGGCGGA TGGTGGTTCCCGCGCCGGGCGGGCTGTGCAGCCGCAGTTGGCCGCCGAGTGCCTCCACCCGGTCGGTGAGGCCGACGAGG CCCGAGCCCCGGCAGGGGGCGGCGCCACCGCGGCCGTCGTCGCGGATGCCGACGTGGAGCCGTCCGTCCCGGGTGGCCAC ATGGACGTCGACGACGGTGGCACCGGAGTGCTTGGCGGCGTTGGTCAGGGCCTCGGAGACGGCGTAGTACGCGGCGGTCT CGACCGGTTCGGGGTGGCGTTCCCCGGTCTGGATGTCGAGCCGGACCGGGATGGCGGAGCGCCGGGCCAGGGCCTTGAGC GCCGGGCGGAGTCCGCCCTCGGCGAGTACCGCCGGGTGGATGCCCCGGGCGACCTCCCGGAGTTCGTCGACGGCGGCGGC CAGCCCGTCGGTCACCTCGTCGAGCTGCCGGATCAGCTCGTCGGCGTCGAGCGGCACCGACAGTTGCACGGTGCGCACCC GCAGCGCCAGGGAGACCAGGCGCTGTTGGGGGCCGTCGTGCAGGTCGCGTTCGATACGGCGGCGGGCGGTGTCGGCGGCG GCGACGATCCGGGCCCGTGACGCGGTGAGGGCCGCCTGCGTCTCCGCGTTGGCGATGGCGGTGGCCACCAGTTCGGTGAA GCCGGCCAGCCGGTCCTCGGTGTCCGACGGCATCGGCTTGTCGTTCATCGACGCCACGCTGAGCGCGCCCCACAGTTGTC CGTCGACGTTGATCGGCATGCACACCGTGGCGCGGAATCCCCACTCCTTGCCGACGACGGAGGCCGGGCCCGAGGACACG GCCGCGTAGTCGTCGATCCGCGCCGGGCAGCCCGACTCGAACACCAGGGTGTGCACATTCCGGCCGCCGGGCGGTACCTG GATACCGGCGGGAAAATCACGGCCGGTCCTGGTCCAGGCGGCGACATACAGGGCGGTTCCGTTGGGCTCGTAACGGCCGA GGACCGCGAAGTCGGCCGAGAGGAGCTGTCCGGCCTCGGCGGCGACCGCGGCGAACACCTCCTTCGGCGGTGCCGCCCGC GCGACCAGGGTCGCCACGCGCCGCAGCGCCGCCTGCTCCTCGGCGGCCCCCCGCAGCTCCACACGTGCCTGGGTGTTCGC GATGGCGGTGGCCACGAGGTCGGTGAAACCGGCCAGCCGGTCCTCGGTGTCGGGCGGCAGCGGTTCCGCGGTCAGCGAGA TCGCCATCATCACGCCCCACAGCCGTCCCTCGACGTTGATCGGCACGCCGACGACCGAAGCGAAGCCGCGCGCCCTGGCG AAGTCGGCGGGTGCCCCGGACGACTCGGCGGCGTCGTCGATCCGGGCCGGCCGCCCCGTCTCGGACACCAGCGTCACCAC GTTCCGGCCGTCGGGGTCCACCCGGGTGCCGATGGGGAAGAGCGGGCCGTGCAGACTTCTGGACCAGCCGCCGACGGCGC TCGCCATGCCGTCCGGATCGAGCCTGATGATTCCGGTCACATCGTTGCCGAGCAGTTCTCCGACTTCGGCGGCGACCGTC GCGAACATCTGTTCCGGTGGGGTGGCCCTGGCCACCAGGGTCGCCACCCGTCGGAGTGCCGCCCGCTCCTCGACGATCTG TTCGCACGACACGACCGCTGCCAGGCCCCCCTACCCGCCCGATGACGCCCGCATACCGGGTATCACGGCACATCAGCATG ACGTCCGCCGTGAACGCCCGTCAACGTGGCCCGCCGGAGTCGGGAACACGCGTCCGGAATCAGCCCCCGGAACGGCGGGA CCGTCTTCCTCCGTCCGGCGCGGGGCACTGCGCCGCGGCGGAATCCGCCCTGACCTCGGGAGTTTGCAGCTAGCTGGAAT CAGCGGTTCGGGTTGGTGGGAAGGGATGTTGGCCGCTGGCGGCGATGCGGAAGCCGATCGTTCCCAGTACTTCTGGGAAG TGCGTCGCGGAGAGTCGGTCCGCTTCCCCGAGTGGGCCGCGACGACGCTGCGGGTTCTCCACGGGGGAGAGATCCGCGAA CCGGCGAAGGAGCTGCCGTGTCGGACGTCTTCGCATCCGAGAAGAGTTCGCCCGGTGTCCGGACCCGCGCGGCAACGTCC CCACCGCGCTCTGTCATCAGCGCCGTCGGCGCCGTCAGCCACGCAGAGAAGATCGGATACGCAGTGTACGAGTGCAGCGA TGAGGTTCGTCACGACGTCCCCGGCCTGCCGGGTCCGTCACCGTCCATCACCGTCCTGGGCTGTCTGGGCGTACGCGCCG ACGGCCGGAAACTGGAGCTGGGCCCTCCGCGTCAGCGGGCCGTTTTCGCCCTGCTGCTCATCAACGCGGGCAGTGTGGTG CCGGTCGACTCGATCGTCTTCCGTATCTGGGGCAACTCACCACCGGGCGCGGTCACCGCGACGCTCCAGTCCTATGTGTC CCGGCTGCGGAAACTCCTGGCCGAGTGTGTGCTCCCGGACGGTTCGACACCCGAACTGCTGCACCAGCCGCCGGGCTACA CCCTCGCGCTCGGCACCGAGCACATCGACGCGAACCGTTTTGAGCAGGCCATCAGGACAGGGCGCCGGCTCTCGCGCGAG GAGCAGCACCAGGAGGCGCGGGCCGTGCTCTGCCAGGCCCTGCTGAGCTGGGGCGGGACACCGTACGAGGAGCTGAGCGC GTACGACTTCGCCGTCCAGGAGGCCAATCGGCTGGAGCAGCTCCGGCTGGGCGCCGTGGAGACATGGGCGCACTGCTGTC TGCGGCTGGGGCGGGACGAGGAGGTGATGGACCAGCTCAAGCCGGAGGTGCAGCGCAATCCGCTGCGGGAGCGGCTGATC GGGCAGCTCATGCAGGCGCAGTACCGGCTGGGGTGCCAGGCGGACGCGCTCAGGACGTACGAGGCGACGCGGCGGGCCCT GGCCGAGGAGCTGGGGACCGATCCGGGCAAGGAGCTGGCGGCGCTGCACGCGGCGATCCTGCGTCAGGACAACGGTCTGG ACCGCGTCGTCCCGGCGTCCGCGCCGCCGTCGGCGGGGGTCGGGCGGGGGGCCGTGACGGTGTCGGTCCCGGCACAGCGG TCGAGGCCGTTGACGCGGCCGGTGGCGGGGCGGGCGCGGGTCCCGGGGGCGATGACGGTGGCGGCGGGCGCGGGGGCGGC CCCCGCGTCCGCCTCCGGCTCCGTTTCCGCGTCCGTTTCCGGCTCCGGCTCCGGCTCCGGCTCCGCTCCTGCGTCGGTTC CCACCTTCTTTCCCGGCTCCGTTTCTGGCTCGGCGTCCGTTGCCGCGTCCGTAGCCGCGCCCGTTTCCGGCCATGTCTCC GGGCCCGGGTCCGCTTTCGGGTCCGTGGCGCTCCACCGGCCGCAGACCCTCCGGGGCGAGCCGGTCCACGGGGGCGCGCA GGGGATGCGCACCGGGCAGGTGTTCCCCACGCTGCCGCCGTTCGTCGGGCGCGGCGACGAGCTGCGCGGTCTGCTGGAGT CCGCGACGTCCGCGTTCCACACCTCGGGGCGGGTGGCGTTCGTCGTCGGCGAGGCGGGCAGCGGCAAGACCCGGCTCCTC TCCGAGTTGGAGCGCTCGGTTCCGGACAGTGTGCGCACCGTCTGGGCGTCCTGTTCGGAGAGTGAGGACCGGCCCGACTA CTGGCCGTGGACGACCGTGCTGCGGCATCTGTACGCGATGTGGCCGGAACGTATGCACGGATTCCCCGGTTGGCTGCGGC GCGCACTCGCGGAACTGCTTCCCGAGGTGGGCCCGGAGCCACAGGGGCCGCACTCCCCCGACGGGGGCGAGGAGAACAGC GGCAACGGGGACGGTGCGGGCGACGGGGACAGCACCCCGGCGCACACCCTCACGCTCGCGCCCGCTCTCGCGCCCCCGCG CTCCAGAGAGGCTCGTTTCACCCTGCACGACGCCGTGTGCCAGGCGCTTCTGCGCACGGTCCGCGAACCCGTGGTGATCA TGCTGGAGGACATGGAGCGGGCCGACGCCCCCTCGCTCGCCCTGCTGCGCCTCCTGGTGGAGCAACTGCGCACCGTCCCC CTGCTGCTCGTGGTCACCACGCGCACCTTCCGGCTCGCGCACGACGCCGAGCTGCGACGGGCCGCCGCCGTGATCCTCCA GTCGACCGGCGCGCGCCGGGTCCTGCTGAACGCCCTGGACGCACGGGCCACCGGGGAACTCGCCGGAGGGATGCTGGGCA AGGCCCCGGACACCCTCCTCGTACGGGCCCTGCACGAGCGCTCCGCCGGGAACCCGTACTTCCTCGTCCAGCTCCTCCGC TCGCTCCGGCAGGGGCTCGCCGCCGCCTGGGAGACGGAGATCCCGGACGAGCTGGCCGGGGTCGTGCTGCAACGGCTGTC GAGCGTGCCGCCCGCCGTGCGCCGGGTGCTCGACATCTGCGCGGTCGTGGAGCGCAGTTGCGAACGGCGTGTGATCGAGA CCGTGCTGCGCCATGAGGGAATCCCGCTGGAGAACGTCCGTACGGCGGTCCGCGGCGGTCTGCTGGAGGAAGACCCCGAC GACCCCGGGCGGCTGAGGTTCGTGCATCCGCTGGTCCGGGAGGCCGTCTGGGACGACCTGGAGAACACCCGTCGGCCCGT GTCCCGTTCCTCCGCGCTCGGGGCGCTGGCCACGGTCTGAGTCCCGGGCCCCGGGGTCCTCGGCGGCGGGCGGCGCTTGC GCGCTCCCCGACGCCGGGCTTGATCCCCCGGGGCAGCCGGACGCGCAGCCGGGTGCAAGGGGCGGTGCCGACACTGGGCG GGCGGCGGGCGTGGCCGGTCGCCGCCCCCCACGGCCCACCGAGGAGCCCCCATTGGACACGTACGCAGCGGATACGTACC CGCGGTCCGGCACCCACCCCGAGCCGCGTCCCGACGCACCTCCCCACGCGCGTCCCGGGACCCGTCCCGGCACCCGTTCC GAGCCGCGCCCGGACCCGGGCGCCGAGGCCGCGTGGCTGCTCGCGGCGGACCGCGCCCATATGTTCCACCCGGTCCTGCC CCGGGGCCGCGAGGACCGCACCGTTCTGGTCTCCGGCCGCGGCTGCACCGTACGGGACACCGAAGGGCGCACCTATCTCG ACGCCTCGTCGGTGCTCGGACTGACCCAGATCGGCCATGGACGTGAGGAGATCGCGCAGGCCGCCGCCGAGCAGATGCGG ACACTCGGTCACTTCCACACCTGGGGCACCATCAGCAACGACAAGGCCATCCGACTGGCCGCGCGCCTCACCGACCTGGC GCCCCAGGGTCTCCAGCGCGTCTACTTCACCAGCGGCGGCGGCGAGGGCGTCGAGATCGCCCTGCGCATGGCCCGTTACT TCCACCACCGCACCGGCAGCCCGGAGCGCACCTGGATCTTGTCGCGCCGCACCGCCTACCACGGCATCGGCTACGGCAGC GGTACGGTGTCGGGCTCGCCCGCCTACCAGGACGGGTTCGGCCCGGTGCTGCCCCATGTGCACCACCTCACGCCGCCCGA CCCGTACCACGCCGAGCTGTACGACGGCGAGGACGTCACGGAGTACTGCCTGCGCGAACTCGCCCGCACCATCGACGAGA TCGGCCCCGGGCGGATCGCCGCGATGATCGGGGAGCCGGTCATGGGCGCGGGCGGCGCCGTCGTCCCGCCGCCGGACTAC TGGCCGCGCGTCGCCGCGCTGCTGCGCTCCCACGGCATCCTGCTGATCCTGGACGAGGTCGTCACCGCGTTCGGCCGCAC GGGGACCTGGTTCGCGGCCGAGCACTTCGGGGTGACCCCCGATCTGCTGGTGACCGCGAAGGGCATCACCTCCGGGTATG TCCCGCACGGGGCGGTGCTCCTGACCGAGGAGGTCGCGGACGCCGTGAACGGGGAGACGGGGTTCCCGATCGGCTTCACC TATACCGGTCACCCCACGGCGTGCGCCGTCGCGCTCGCCAATCTCGACATCATCGAACGGGAAGGGCTGCTGGAGAACGC GGTGAAGGTGGGCGACCACCTCGCCGGGCGGCTGGCGGCCCTGCGCGGGCTGCCCGCCGTGGGGGACGTCCGGCAACTGG GCATGATGCTCGCCGTCGAGCTGGTGTCGGACAAGACGGCCCGCACCCCGCTGCCGGGCGGCACCCTCGGGGTCGTGGAC GCGCTGCGCGAGGACGCGGGCGTCATCGTCCGGGCCACGCCGCGCTCCCTGGTCCTCAATCCGGCGCTCGTGATGGACCG GGCCACGGCGGACGAGGTGGCGGACGGGCTGGACTCGGTGCTGCGGCGGCTGGCACCCGACGGGCGGATCGGCGCGGCCC CCCGGCGGGGGTGACGAGACCGCGGGCCGCCACCCGCGGGGGGCGCCGGGTCGGCACAGCGGCCGACCCGGCGCCTTCCC CGTTTCCCGGCGCCTTTTCCGTGCCCCGGCGCCGTTCCCGTGGCCCCTGCCCCTGCCCCTGCTCGGGCGCTCCTCCCTCC GCTGTGGCGCCGTTCCCGTTCCAGCGCGCTGTCGAGCCGCCGCCAAGCGCCCCGTGCCACGGTGGGAGACCGCCGCCCGA CGGGGCGCGCGGAGCCCGGCAAGCCGAAGGGAAGTCCCGTCCGATGCGTGCCTCTTCGCCCAGAGGGTTCCGCGTGCACC ACGGTCACGCCGGGATCAGGGGGTCCCACGCGGACCTCGCCGTCATCGCCTCCGACGTTCCCGCGGCGGTCGGCGCGGTG TTCACCCGTTCGCGGTTCGCCGCGCCGAGTGTGCTGCTCAGCCGGGACGCGGTCGCCGACGGGATCGCCCGGGGCGTGGT GGTGCTGTCCGGCAACGCCAACGCCGGGACGGGCCCGCGGGGGTACGAGGACGCCGCGGAGGTGCGCCATCTGGTGGCCG GGATCGTCGACTGCGACGAGAGGGATGTGCTGATCGCCTCCACGGGACCCGTCGGCGAGCGGTATCCGATGTCCCGTGTC CGGGCCCATCTGCGGGCGGTGCGCGGGCCCTTACCGGGTGCCGACTTCGACGGCGCGGCGGCGGCCGTGCTGGGCACCGC GGGCGCCCGTCCCACGATCCGGCGGGCGCGGTGCGGCGACGCGACGCTGATCGGTGTCGCCAAGGGCCCGGGTACGGGCC CGGCGGAGCAGGACGACCGGTCGACGCTGGCGTTCTTCTGCACGGACGCCCAGGTGAGCCCCGTCGTCCTCGACGACATC TTCCGCCGGGTCGCGGACCGCGCCTTCCACGGGCTGGGCTTCGGCGCCGACGCCTCCACCGGCGACACGGCGGCCGTTCT CGCCAACGGGCTCGCGGGCCGGGTGGACCTCGTCGCGTTCGAACAGGTCCTGGGCGCGCTGGCGCTGGACCTGGTCAGGG ACGTCGTCCGGGACAGCGGCTGCGGCGGCGCCCTGGTCACGGTGCGGGTCACCGGGGCCCACGACACCGAGCAGGCCGGG CGCGTGGGCCGGGCGGTGGTCGACGCGCCGTCGCTGAGGGCCGCGGTGCACGGCCCGGCACCCGACTGGGCGCCGGTCGC CGCCGTGGCGGGTGGACACGGGGACGAAGGCCCCGGCCGGTCTCCCGGGCGGATCACGATCCGGGTCGGCGGCCGGGAGG TCTTCCCCGCCCCCCGCGACCGGGCCCGCCCGGACGCCGTCACCGCGTATCCGCACGGCGGCGAGGTGACCGTCCATATC GACCTCGGTGTCCCGGGCCGGGCGCCCGGCGCGTTCACGGTCCACGGCTGCGACCTCCTGGCGGGGTACCCGCGCCTCGG CGCCGGCCGGGCCGTCTGAACGGGCGCTCCCGGGCGGACGGCGACCGCGAGGGCGCGGGAGCGCAGGGAACACGGGAGCG GGCCCGGTGGTCGATCGGCCACCGGGCCCGCTCCCGTCGTTCCGTCCGCTGTCCCCGGCCGCCCTACCCCCACCGCTGCC CGGCGAAGTCCACGGCGCTCTCGGCGTCCACCGCGTCCACCGCGTTCTCGGCGTTCTCGGCGTCGTCCGCCGCCGCCCCC GGTGGCAGGGGAGAGTCCACCGGTGCCGACGCGGGCGACGTGGTGGCGCGGGCGTACTGGTAGAGCAGTTCGGCCCCGAT CTCCGCCGCCAGCAGGGAGGTGATCCCCGACGGGTCGTACGCCGGGGACACCTCGACCACGTCGAAGCCGACGGGCCTGA GCTGCCCGACCACGTCGAGCAGGGTCAGCACCTCGCGCGAGGACAGCCCGCCGGGGGCCGGTGTGCCGGTGCCCGGGGCG TACGCCGGGTCGACGACGTCGATGTCGACGGAGACGTACAGCGGCAGGCCGCCGACGGTGCGCCGGATCTGCTCGGCGAT GCCGCGCGGTGAGCGCCGGGTGAAGTCGGCGGCGGTGACGATGCTGACGCCGTGCCCGCGCGCGTAGTCCAGGGAGTCGG GCCGCGGATTGTGGCCGCGGATGCCGACCTGGACCAGGCGCTCCGGGTCCACCAGGCCCTCTTCGATGGCCCAGCGGAAG GGGGTGCCGTGGTGGTAGGTGCCGCCGTAGACGGGTGGGTTGGTGTCGCTGTGCGCGTCCAGGTGCAGGACGGCGACCCG GCCGTGGCGGGCGTGCACGGCGCGCAGGGCGGCCAGGGAGAGCGAGTGGTCCCCGCCCAGCATCAGGAACGCGTCGTTGC GTTCCAGGAGCCGGGTCAGGGCGACCGTCGCGGTGTCCATCGCCAGGTCCATCGAGAAGGGGCTGAGGTCGATGTCGCCC CCGTCGACCACGTCGATCCGGTCGAAGACCCCTGGGCCCCGGTCGATGCCGACGCCGTGGATCAGGCTGGACTCGTGCCG GATGGCGCGCGGCGCGAACCGCGCGCCGGGCCGGTAGCTGGTGCCTCCGTCGTACGGGGCGCCGACGACCACCACGTCAT GGCCGATCGGGTCGGGCCGGTGGCGCAGCCGCATGAAGGTCGCCGGTTGGGCGTAGCGCGGGGAGACGGCGGTGGACACC CTGGCCGTTCCCCGCGCACCCGGCCCTGCTCCCGTTCCCGTACCGACGCCCGGCCACCCCGTGCGGGCTCCCGTTCCCGT GCCGACCCCCGTTCCCGAACGGGCTCCCGTTCCCGCGTGGAATCCCGTTCCCGCGCCCGCGGCGCCGTCCGGGCCGCGGC TGCCCCTCCCTCCGAGACCGCTCCTGCCGTTCCTGCGGCCGTTGCCGCTCTGCGGGCCGGTGCCCGCGCCCACGCCCGCT GCACCGTCCGCGCCGCCGCCGGTGCCGTTGCCGCCGCCGGTGCCGTTCTGGCCACCGGTGCCGTTCTGGCCGCTCATACG ACCACCCGGCCCTGGAGCCTGAGCCTGCGCACCGCGTCGACGGAGCGCCGCACCGTCTCGCCGAAGTCCACGTCCTCCGG CGGCACCGTGTCGATGACCACCGCGTCGTACAGGCGCCGTGCCATGGCGCCCTTGACGGCCGTCACCTCGTCGCGCCGGA TCCCTTCGGCGAGGAGCAGTCCGGTCCACGCGCTGGTGGTGCCGGACCCCTCGTGGATGCCCAGCTTGGGGCGGGCCACG GTCTCGGCGGGCAGCAGGCCGGAGAGGGCCTGCCGCAACACCCACTTGTCGGTGCCCCGCCGGCGTTTGAGCCCGGGTTC GAGGGAGACCAGCGCGTCCAGGACCGCGCGGTCCCAGTACGGGTGGGTGGTCCACTTCCCGGCGATGCCCGCGAGGACGG GGGACATCTCGTTGAGGCCGTCGAAGCCCGCCATGTCGCCCGCGATCTCGTCGTCGAGGGACCAGAGCGAGGCCGTGCGC CGGTGCATACCGCCGAGCGGGATGTCGGCGCCGTACCCGGTGAGGATGCGGAGCGGCCCGGTGTCGAGCCGCCGGTAGAG GGCGACGAGCGGCAGCAGGTACTCCAGGACCGTGGGGTCGGTGATCTCCGCGGCGGCGACCGCCCAGGGCAGTTCCCTGA CGAGTTCGGCCGAGTGGAGCCGGATCTCGCTGTGCGCGGTGCCCAGGTGGACGGCGACCGAGCGGGCCGCGTCGAACTCG TCGGACACCTCGGTGCCCATCGACACGGACCGTGTCCCGGGTGCCAGGGCCGCCGTGTGGGCGGCGACTCCCCCGGAGTC GATGCCGCCGGACAGGACGACGGTGGGGGCCGCCTCCCCGCCGCGCAGCCGGGTGCGGACCGCCGTGGCGAGGCGTTCGC CGACCAGGTCCACCGCCTCCCGTTCGCCGGGCAGCGCCCGGGAGAGCGGGGGTGTCCAGGTGCGGACCGCCCTGGCGGTG ATGTCGGAGCCGCCGACTCCGTGCAGCAGGAGGGCGGTCCCGGCGGGGACCCGGCAGACGCCCGCCGCCCCCGGCGCGGT GTGGGTGCCGGACAGGCCCAGCGGCCGGCCCGGCTCGTGCGCCAGGGTCTTCGCCTCGGTGGCGGCGCTCAGCCCCGTCA CGTCGGCGCGCAGCCACAGCGGTACCGAACCGGCGTGGTCGGTGGCCGCGACGGTCGCGCCGGTGGAGGCGTCGGTGAGC AGTGCGGCGAACCGTCCGTTCAGGAGCCGGAAGGCCCCGGGGCCCCAGCGCCGCCAGGCGGCCAGCAGCAGTTCGGCGTC GCCGAGGGCGGCAGAGGAGCCGCCGAGCGCTCCGGTCAGCTCGGCGCGGTTGTACAGCTCGCCCGCCAGGAGCAGCCGGA CCTGGCCGTCGGCGACCAGGACGGGCGGACGGCCCAGGGTCACGGCCGTTCCGCTCCAGAGCGGGTACGCGGTGCCGTCG TGCACGGGGACATGGGTCCCGCGGACGGCGAAGCGGGGTGCGCTGCCGGGTTCGGAGTGACCGCCGGGGCCGCCGCCGGG GCGGCCCTCGGTGCCGATGCGCACCCGGAATCCGTACACGAGGTCGGGGCCGGGCATGGTGAACTCGTCCTCCACGGTGG TCAGATGGCCAGGGCGGCGAAACCGCCGGACTGGAAGTCGTAGGCCACCGGTACCTCGATCAGGAACGGGCGGCCGAGTC CGGCGCCCTTGGTGAGGGCGGCGAGCAGCGAGGTGCGGTCGGTGGCGCGGACGGCCTCGCAGCCGTTGGCCTCGGCGAGC TGGACGAAGTCGACGCTTCCGAAGCCGACGGCGGGGGCGTGGGAGCGCTGGTGTCCGAGGTTCTGGTACAGCTCGATCAG GCCGTTGCGGTCGTTGTTGACGACGACCATGACGATCGGCAGGCCCAGGCGCACGGCCGTCTCGATGTCGGCGCTGTTGG AGTGGAAGCCGCCGTCGCCCGCGATGAGGAAGACGGGCTCGCCGGGCCGGGCGATCTGGGCGGCCATGGCGGCGGGCAGT CCGTAGCCGAAGCTGGAGCAGCCCGCGGAGGTGAGGAATCCGTACGGCTGGTCGGACTTGGCGAAGAGCACGCCGTAGTG GCGGAAGAAGCCGATGTCGCTGACGAAGGTGCCGTTGTCGAGGACGGAGTTCATGCAGTCGATCACCTGGTGGACCCGCA TGCCGTCCTCGTACTCGGTGGGGTCGGCGAGGAATTCGGCGACGCGGGCGCGCAGGGCGCTGAGGTCGTGCCGGGTCTTG GGGGCGAGGCCCGAGGTCGCGTCGTCGAGCGCGGTGACGAATTCGGCGACGTTGGTGACGATGTCGATGTCGGCGCGGAA CAGCTCCGGGATCGGGTTGACCTCGGGGGCGACCCGGACCGTGGTCTTGGCCCGGCCCCGCGTCCACATGGAGGGGCGCA GGTCCTCGGCGTAGTCGTAGCCGATCGCCAGGAGGAGGTCGGCGGGGCCGAAGATCTCGTCGAGGGCCGGGTGGCCGAGA ATGCCGTCCATGTAGCCGCTGATGGCGCCGTAGTTGAGCGGGTGGTCGTGCGGCAGGACGCCCTTGGCGGTGTAGGTGGT GACGACGGGGATGTTCAGCCGCTCGGCGAGGGCGCGCAGGGCGTCGACGGCCCCGGCGCGGATGACGGCGCTACCGACGA CGAGGAGGGGGTTCTCGGCCTCGCGCACCAGCTCAGCGGCCTCGTCGAGGCGGGCGCGCCAGTCGGCGTCCAGGGCGTGG GTGGCGGTGGCCCGGACCAGGGGGGCGTCGGTGGGGGTGCCGTTCAGCTCGGCGCCGAGGAGGTCGACCGGCAGGCTGAT GAAGCTGGGACCCACGGGCTCGATCCGGCTGTTGAGGACGGCGCTGTCGACGAGGTTGACGATGTCCTCGCCGCGTTCGA GCTGGACGCTGAACTTGGTCAGCGGGCCCATCACGGCGGTGCTGTCCAGGCACTGGTGGGTGACGTTGGGGTAGCAGTCG TACGACTCGGACTGCGCGGCCAGCGCGATGACCGAGCTGCGGTCCAGGGCGGAGGTGGCGACGCCGGTGGCCAGGTTGGT CATGCCGGGGCCCAGGGTCGCGAAGCACGCCTGGGGGCGGTTGGTGATCCGGGCGAGGACGTCCGCCATCACCCCGGCGG TGAACTCGTGCCGGGTCAGGACGAAGTCGAGTCCTTCGACCTCGTCGAAGAGAATGGCGGACGCCTCCCGGCCGACGACG CCGAATACATGGTCGACACCGTACTGGTGAAGACGTTCCAGCATGGCTTTCGCGGTCGTGGTGGCCATGGAGATCTCCTT CGCATCGGACGGGCGCCGGGATGGCGCCCCGGAAAACGCGGCACCGGGCGGTGCGCACCGGGTGGCGCACACCGTGGGTG GTGGCGTTGCCACTGTGCGGATCGCCTCTTGGCGGCGGTCGGACGCCCGGCTTGGACAGAATGGGCAAGGCGCGTTCAAG GCATGGCGTCCATCGTCCTCGTGGCGCTTTTCGTGAAATCCGTCCGGCGCCGACGGTCTCCATCCGATTCCGTCCCCTTC CGTCCACCGATCCGAGGAGAATCCATGGATGTCCTGGCCGCGTTGGAGCGCAAGCCCAGCCTGAATCTTTTCCCCATCGA GAACCGGCTGTCGCCGCGCGCCAGTGCCGCGCTGGCCACCGACGCCGTCAACCGCTATCCGTACTCCGAGACCCCGGTGG CCGTCTACGGCGATGTCACGGGGCTGGCCGAGGTGTACGCGTACTGCGAGGACCTGGCCAAGCGCTTCTTCGGGGCGCGC CACGCCGGTGTGCAGTTCCTGTCCGGTCTGCACACCATGCACACCGTGCTGACCGCCCTGACCCCGCCCGGCGGGCGCGT CCTGGTCCTCGCGCCGGAGGACGGCGGCCACTACGCCACGGTGACGATCTGCCGGGGCTTCGGCTACGAGGTCGAGTTCT TACCTTCGACCGCCGGACACCTGGAGATCGACT
SEQ ID NO: 17 cvm cluster
GGTACCGGCATCCGACCCAGGCCCCGGGCGCAGGACCCGGAGGCAGGCACCGGCACACCC CGGCCGGGCGGCCCGGCTCCCGGCGGTCGGTGTCCGGCGACCCGCAATCGGCAGCCGCCC CAGGCCCGGGACAGGAGCCCGGCTCAAGGCACCGGCCCTGCGCACCCGCTGAGGCGGCAG GTTCCTGACAGCCGGCATCCGCCAGTCGGCGCGGGGCAGCCGCCCCAGGCGCCCGGCCCG GCACACCCGTGCGAGCGCCCGGCTCCCGGCGGTCGGTGCCCCGGAGGCGGCGACCGGCAG CCGGACACGGCCCCGCTCGGGGCGCGGCCCAGGGCACAGGCCCTGGGCACCCGCTCGGAC GCCCGTTCGGACAGCAGGCCCGTGGGAAGCCGCCGGTCAGGCCCGCAGGCAGCCACCGGT CGGCGGGCGGATCAGGTGTTGGCGGGGGACTCGTCCGGGAAGATCTTTGTGACGACGGTC CCGTCCTCGGTCAGATAGCCGTGCAGCATCCCGGGGCTGCTGTGCGGCGCGTCGAAGTCG
CCCCGGGGGTCGAGGGCGATCACGCCGCCCTGCCCGCCGAGCCGGGGCAGGCGCTTGACG ATCACCTCGTAAGCGGCGGACGCCACGCCGAGCCCCTTGAACTCGATCAGATGGGAGAGG GTCGAGGTCGCCGCGCCCCGGATGAACACCTCACCGGCGCCGGTGGCGCTCGCGGCGACG GTCCGGTTGTCGGCGTAGGTCCCGGCCCCGATCAGCGGGGAGTCGCCGATCCGGCCGGGG AGCTTGTTGGTGAGCCCGCCGGTGGAGGTGGCCGCCGCGAGATCGCCGCGCCGGTCGAGG GCCACCGCGCCCACCGTCCCCGTCGACTGCGCGTCGGCCAGTGCCTCCGGGGCCCTCCGG GCGGCGGGATCGCCCGCCTCGGTCTCCTTCGCGCGCAGCAGCGCGTCCCAGCGGGCCTGG GTCCAGTAGTAGTCCTGGGTGACGGTGCGCAGCCCGTGCCGGGCGCCGAAGTCGTCGGCG CCCTCGCCGGAGAGGAGGACGTGCTTCGACTTCTCCAGCACCAGCCGGGCGCCCTCGACC GGGTTGCGCAGGGAGGTGACCCCGGCGACCGCTCCCGCCTTCAGATCGGAGCCCCGCATC ACGGAGGCGTCCAGCTCATGCCCGGCGTCGGCGGTGAAGACGGCGCCCTTGCCCGCGTTG AACAGCGGGTTGTCCTCCAGTTCGCGGACGGCGGCCTCGACCGCGTCCAGGCTGTCCCCG CCGCGCGCGAGCACCCGCTGTCCGGCGCGGAGCGCTGCGGCGAGCCCGTCCCGGTACGCC TTCTCCCGTTCCGGGCCGGTCGTCTCCCGGTCCAGGGCGGCTCCGGCCCCGCCGTGGACG GCGATGACCACGTCACGGGCGTCCGGCCGGGGCTTCCCCGGCGCGCTCCCCCGTTCCTTC TTCTCCTCCCGCGCCTGCTGCTCCTGCTTCTGTTGCGTCGTGTGGGCCGCCGCGGTGGGT CCATGGCCGCCCGAGGCCCCGGGTACGACGATGAGCGTGGTCGTCAGCACCGCGGCGGCG AGCAGGGAGGACGCCAGCCAGGCGGTGGCGGGGCGGTGGGGCATCGGGCACTCCTCGGGA CGGGGGTGAGAGACGCTCCGGCCGACTGTACTGACATGCCCATGCCCCCTCTAGTGCCCC GGAGCCGCCTTCCGCCCTCCCCGCCGCCCGGCGGCGCCCGCCCGGCGCGCTCAGTCCAGG GCCAGGTCCTCCGGGGCGGAGCGGGCGAGTCCGGCGAGTGTGCCGAGCGCCCGGGTCAGT TCGTCCGCCGACGGCGACGCCAGGCCCAGCCGGACCGCGTGCGGTGTACGGCCCTGCCCG GCGCAGAACGCGGCGGCGGGCGTCACCCCGATCCCGTGCCGCGCGGCGGCGGCGACGAAG GTGTCGGCGCGCCAGGGGCGGGGCAGCACCCACCAGCAGTGGTACGAGCCGGGGTCGCCC GACACGGCGAAGCCGTCGAGCGCGCGCCGGGCGATCTCCTGCCGTACGCCCGCGTCCCGC CGCTTGGCGCGTACCAGCGCGTCGACCGTGCCGTCGGTCTGCCAGCGGACCGCCGCCTCC AGCGCGAACCGCGCGGGGCCGAGACCGCCGGAGCGCAGCGCGGCGCCGACCGCTCCGTCG AGCCCCGGGGGCACCACCGCGAACCCCAGGGTCAGCCCGGGGGCGAGCCGCTTGGAGAGG CTGTCGACGAGCACCGTCCGCCCGGGGGCGACCGCCGCGAGCGGAGCCGTGCCCTCCCGC AGGAAGCCCCAGACGGCGTCCTCGACCGCGGGAAGGTCCAGCCGCTCCAGGACCGCGGCG AGCTGGGCGAGACGCCCGTCCGACAGGGTGAGGGAGAGCGGGTTGTGCAGGGTGGGCTGG ACATAGACCGCCCGGAGCGGAGCGCTCCGGTTGGCCTCGTCCAGCGCCTCCGGAATCACC CCGTCCGCGTCCATGGCGAGGGGGACGAGCGTGATGCCGAGCCGGGCCGCGATCGCCTTG ACCACGGGGTAGGTCAGCTCCTCGACCCCCAGTCGGCCCCCCGGCGGCACCAGCGCGCCG AGCACGGCGGAGAGTGCCTGCCGACCGTTGCCCGCGAACAGCACCCGCCGGGGGTCCGGC CGCCAGCCGCCCCGGGCGAGCAGCCCGGCGGCGGCCTCGCGCGCCTCGGGGGTCCCGGCG GCACCGGCCGGCCGGAGCACGGACTCCAGGACATCGGGCCGCAGCAGCCCGCCGAGCCCG GTGGCCAGCAGCGCGGCCTGCTCGGGGACGACGGGGTGGTTCAGCTCCAGGTCGATCCGG CTTCCGGCGGGCTCGGAGAGCGCGGGGCCGACGCCCGCCCGCGCCGCGCGGACATAGGTG CCGCGCCCCACCTCGCCGACGGTGAGCCCTCTGCGGGCCAGCTCCCGGTAGACCCGGGCG GCGGTGGAGTCGGCGATGCCGCACCCGCGGGCGAACTCCCGCTGCGGCGGAAGCCGGTCC CCGGGGCGCAGCCCGCCCGTCCTGATCTCCTCGGCGACCGCGTCGGCCACCTGCCGGTAG TCCTTCATCTCCCGTACCTCCCCTGTCCGGTGGACCGCTTCCCGCCCGGCCCCGCCGACC GTGAAACGGAAGCACCCCGTTCCGGAGCTCGAGCTCCCCGTCCGGAAGCTCCCCGTCCGG AAGCTCCCCGTTCCAGAATTGCACCGAGAGCAATATTCCCTATTGCACCGATCAAAACAC CGATCTACGCTCGGAATTGCCTCACACAGACCGTCGACGCATCTGCCGCACACCGGTACT GACGCCCCGTCGGACCGCACCCGCGCGGAGCCGTCGCCCCGCCCGCCCCGTTCGCGCACA GGAGAGAGAAGGAGATGGTGGAGACCAGCGCACTCGCCGGTGTGGTGATGGTCGCCCTCG GAATGGTCCTCACCCCGGGACCGAACATGATCTATCTCGTCTCCCGCAGCATCACCCAGG GCCGACGTGCGGGGATCATCTCGCTGGGCGGTGTGGCCCTCGGTTTTCTGGTCTATCTGC TCGCCGCGAATCTCGGCCTGTCGGTGATCTTCGTCGCCGTGCCGGAGTTGTATGTCGCGG TCAAACTGGCCGGTGCGGCCTATCTGGCATATCTCGCCTGGAACGCCCTGCGGCCCGGTG GCGTGAATGTGTTCTCCCCCGAGGAGGTTCCGCACGACTCCCCGAGCAGGCTGTTCACCA TGGGGCTGATGACGAACATCCTCAACCCCAAGATCGCCGTCATGTATCTCGCACTCATCC CGCAGTTCGTCGACCCGAACGCGGACCGTGTCCTGTTCCAGGGGCTGATTCTCGGCGGTC TCCAGATCGCGGTGAGCGTCGCGGTCAATCTCGCGATCGTGCTGGCGGCCGGAGCCATCG CCGCCTTTCTCGGCCGCCACCCCTTCTGGCTCAGGGTTCAGCGCCGCGTGATGGGCGCGG CGCTCGGTACGCTCGCGGTCTCCCTGGCCCTCGACACCTCCGCCCCCGCCGCACCCGTCT CCTGAGGCCGCCGGACCGGGAGCCGACGCGAAGGCACCCCTGGGCAACCGTTCGGAGAGC TTATCCGTTACCCCATGAATCCCGATATAAGTGCATTGGCCACTTACCCATGCATGGAAC AGGCCAACCTGACCAAAAAATGAGCCCTCCCCACCCGGAATAGATGCTTCCCAGTGTGAA GAAATTTCATAGCGGGAGCGTCTGCCGAACAGGACGGCCCATACGCCGCAAGGCAGAACG GACATCGCCGCCCGCCCGGGTCCAGAAAATTCGGAGGACACATCGGACGACCGTCTCCGC ATCGGCGTCAACTCCCGATTACAGAGAATATTGAGTACGTATCAACCGGGCCTTGATCTA CTCAGCCTCCATTGTTCTCTCCAGTCGGGATGTGCAATGAAGTACGACATAACCCCACCA TCCGGCCTTCGGTTCGACCTCCTCGGCCCGTTGACCGTGACCGCCGGCGAGCAACCCGTG GACCTGGGCGCGCCACGGCAGCGCGCCCTGCTCGCCCTGCTGCTCATCGATGTCGGCAAC GTGGTCCCGCTGCCGGTCATGACCGCGTCGATCTGGGGGGCCGACCCACCGTCCCGGGTC CGGGGGACGCTCCAGGCTTATGTGTCCCGACTGCGGAAACTCCTGCACCGCCATGACCGT TCCCTTCGCCTTGTCCACCAGCTCCAGGGGTATCTCCTCGAAGTGGATTCGGCGAAGGTG GACGCCGTGGTTTTCGAGACACGTGTCAGGGAGTGCCGGGAATTGAGCAGGGCCCGGAAC CCCGAGGCCACCCGGGCCGTGGCCTGGTCCGCCCTGGAGATGTGGAAGGGCACACCCATG GGCGAGCTGCATGATTATGAATTTGTGGCGGCGGAGGCCGACCGGCTGGAAGGAATCCGG TTACGCGCGCTGGAGACCTGGTCCCAGGCGTGTCTCGATCTCCAGCACTATGAAGAGGTT GCATTTCAGCTCGGCGAGGAGATCCACCGCAATCCGGAACTGGAACGGCTGGGCGGTCTC TTCATGCGGGCCCAGTATCATTCCGGACGGTCGGCGGAAGCCCTGTTGACGTATGAACGT ATGCGTACCGCGGTGGCGGAGAATCTGGGGGCCGATATCAGTCCGGAGCTCCAGGAACTC CATGGAAAGATTCTGCGCCAGGAACTCACGGAGACACCCGCCGCGCGATCGACGGCCTCC CTCACACGGGCGGCGGGCCCGCACGGGCCCCCGCCCCTGGCCGAAACCGGCACCCCCGCC GCACCCGCGGACATGGCCGAAACCACGGTGGCGGAGGAAAGCGCCGCGCCCCCCGCCCCG GCGGCGCCCGGGACCCCGCCCCCCATGCCGTCCCCCGTACCGCTCCCCCATCCGTCAGGG GCCGTCCCGCCGGTCACCCCGGTGCCTCCCCCGGTCCCCCGCTCGGCCCTCCGTTCAGCG GCACCCGCCGAGACCGAGGACCCGGAACCGGCGCCGCCCCCTCCCCCTCCGCCGGGCGGC CGACTCATCGGCCGCCGCGCCGAACTGCGCAGGCTGCGGCTGGTGCTGACGAAGACCCGC GCGGGCCACGGCCATGTCCTGCTGGTCTGCGGCGAACAGGGCATCGGGAAGACCCGGCTC CTGGAGCACACCGAGCACACCCTGGCCGCGGGCGCGTTCCGGGTGGTCCGTTCGCACTGC GTCGCCACCCTCCCGGCACCGGGCTACTGGCCCTGGGAGCACCTCGTACGCCAGCTCGAC CCGGACAGCGGCCTCGGTGACGACGGCGACGCCGACCCCGTCGCCCAGGCCGAGTGGCTG CCGGAACACCACCTCACCCACCAGATGCGGATCTGCCGGACGGTGCTCGCCGCGGCGCGG CGGACCCCGCTCCTGTTGATCCTGGAGGATCTGCACCTCGCCCACGCGCCGGTCCTGGAT GTGCTCCAGCTCCTGGTCAAACAGATCGGCCAGGCCCCCGTCATGGTCGTCGCCACCCTG CGCGAGCACGATCTCGCCCGGGACCCCGCCGTCCGCCGGGCCGTGGGCCGCATCCTCCAG GCGGGCAACACCGGCACCCTCCGGCTGGACGGGCTCACCGAGGAGCAGAGCCGGGAGCTG ATCGTCTCGGTCGCGGGGGCCCCGTTCGCGCCCCATGACGCCCAACGGCTCCAGCGCGCC TCGGGCGGCAACCCGTTTCTGCTGCTCAGCATGGTCACAGGGGAGGACGGCACCCAGGAG TGGGCACGGCCGTGCGTCCCGTTCGAGGTGCGCGAGGTGCTGCACGAGCGGCTGAGCGAA TGCTCCCCGTCCACCCAGGACGTGCTCACGCTCTGCGCCGTGCTCGGCATGAGCGTGCGC CGACCGCTGCTCACCGACATCATGTCCACGCTCGACATCCCGCACACCGCGCTCGACGAG GCGCTCGGCACGGGGCTGCTGCGCCACGACCGGAACACCGACGGAATGGTCCACTTCGCC CATGGGCTGACCCGGGACTTCCTGCTCGACGACACCCCGCCGGTCACCCGCGCCCGCTGG CACCACCGGGTCGCCGCCACCCTCGCCCTGCGCTTCCAGCAGGGCGACGACCACGCCGAG ATCCGCCGCCACTGTCTGGCCGCGGCCCGTCTGCTCGGCGCCCGCGCGGGGGTGCGCCCC CTGCTGGCGCTGGCCGACCGGGAGCAGTCCCGCTTCTCCCACGCGGAGGCGCTGCGCTGG CTGGAGAGCGCGGTCGCGGTCGTCGCGGCGCTGCCCCGGGACCAGCCGGTGTCCGCCGTC GAACTCCAGTTGCGCAAACGGATGATGGCGCTGCACGCGCTGATGGACGGCTATGGATCG GCCCGCGTCGAGACGTTCCTCTCCCAGGTCACCCAGTGGGAACACGTCTTCGACAACACC CAGCCCACCGGGCTGCTGCACGTCCAGGCGCTGAGCGCGCTCACCACGGGCCGCCATGAG CAGGCGGCGGAGCTGGCCGGGCTGCTGCACGAGCTGGCCGACCACGGCGGCGGACCGGAG GCCCGGTCGGCGGCCTGCTATGTGGACGGCGTCACCCTGTATGTGGGCGGACGGGTCGAC GAAGCCCTCGCCGCGCTCGCCCAGGGCACCGAGATCACGGACGCCCTCCTGGCCGGACAC CGCAGGACCGCCGCCCCGCACGGCGGCGGGCACCTCCAGGACCGGCGTATCGACTTCCGC GCCTATCTGGCGCTCGGCgACTGTCTCAGCGGCGACCGGATTCAGACCCAGCGCTACCGG ACGGAACTCCTCCACCTCACCCAGTCGGAACGGTACGACCGGCCGTGGGACCGGGCCTTC GCCCGCTATGTGGACGCGCTCATCGCCGTCACGGAGTGCGATGTCCAGGGGGTGTGGCTG GCCGCGCGGGCGGGGCTCGACCTCGCCGCCCGCTGCCAGCTCCCGTTCTGGCAGCGGATG CTCGCCGTCCCCCTCGGCTGGGCCGAGGTCCACCAGGGGGCGCACGACAAGGGGCTGGCC CGGATGCGGGAGGCGCTGCACGAGGCGGCCCGGCACCGGACCCTGCTGCGCCGTACGCTC CACCTCGGGCTGCTCGCCGACGCCCTCCAGTACACGGGCGCCCGGGAACAGGCCCGGCGC ACGATGTCCTCCGCCGTACGGGAGATCGAGCGCCGCGGCGAGTACTTCTGTCTCCGGCCG CAGTGGCCCTGGGCCCGGCTCCTCCACAGCCACGGCACCTCCGCCGCGGCGGAGCACCGG GTCGTCCACGGCAGGCACTGACCCGGGGCCGGCCGGAGCCGGGCCCGTACGGTACGGGTC CGGCTCCGGACCCGGCGGCCCGGAGCCGGGCGGGGCGGGGCGGCCCGACGGTTCCGGGGC CGGCGGTTGTGGGAGGGGGCGGCCCCCGATCGCTCAGACCGGGCAGACGGCGGACCGCCG CCCCGCCCGGCCCGAGCCGCCGCCCCCGGCCCAGTGCCCGTAGTCGCCCCGCAGGAAGAC CAGGGGCGAACCCTCGCGGATCACCCCGAGGTCGCGCACCGCCCCGGTGACGAACCAGTG GTCGCCCGCCTCCGTCTCCCCCGCCACCTCGCAGTCGAACCACGCGAGCGCGTCGAGCAG GACGGGGGAGCCGGTGGCCGTCGTCCGGTACGGCACCTCCCAGCGCCCCGGATCGCCCCC GGCGAAACTCCGGCAGACCGGGCCCTGATCCGCGCCGAGCACATTGACGCAGAAACGCCC GGCCGCCCGGAGCCGCGGCCAGGTCGTCGACGACCTGGCCGGGAGGAAACCCACCAGCAC CGGATCGAGCGACACCGAGGTGAACGTCCCCACCACCATGGCGGGCGGCGGCTGCCCCGG AGCCTCGGCCGGACCGGTGACCAGGACCACCCCGGTGGGATAGTGGCCCGCCACCCGGCG CAGCAGACTCCCGGACACGGACCCGTGGGTGTGCGCGGAAAGGCCCGGAGGCCGGGTCAC AGCCACGGGTAACGCGCGGTGTCCTTGCCCGCGTAATCGGGGTCCAGATAGACGAAGGCC CGGTGGACGAGGAAGTCCCGCACCTCGTAGACCGTGCACCAGCGCCCGGCGGCCCACTCG GGGTCACCCGCCCGCCACGGCCCGTCCCGGTGCTCACCGTGGGTGGTGCCCTCCGCGGCG AGGAGTTCGGTCCCGGTCAGAATCCAGTTGACGGACCACAGATGGTGGGTGATCGAGCGG ATGGTGCCCCCGAGGTCGTCGAAGAGCCGGGCGATCTCGGACTTGCCCCGGGCCAGACCC CACTTGGGGAAGAAGAAGACCGCGTCCTCGGCGAAGTAGTCGATCGCGGGGGTGCCGTCG CTGCCGACGCCGCCGTTGTCGAACGCCTTGAAGTACGCGGTGATGACCGCCTTGCGCTGC TCGTCCGTCATACCGGCCGATGCCACGGACATGAAACGACCTCCAGAGATTCCGGGTGGC TGTGCTGGGGCTGCGGAAGGGGTGTCCCCCGCGAAGGACGGCGGACGCCGCGGACGCCGC GGCCGTCTCCCCGGCGGACGGGTCCCAGCGTCCTGGAGAGGGCTTGGCGGCGGCTTGACG CCGTGCTGTCCCGCGGCTTGCGGAACGCGAAGTACCGGCCAGCGTACGGGCGTTGCACCG GACGTGTACGCCGGTCGGGACCCCTCGTACCCCCGGAGCCGGCCGACCCCGGCGGCTCCG GGGGTACGGACGCGCCGGACCGGCCCGAGCGAGCCGGACGGGTCGGACGGTGCGCGTGGT TCCGGTGTGTCGGACAGCTCGGACGGACCGGACGGTGCGCGTGGTTCCGGTGTGTCGGAC AGCTCGGACGGGTCGGACGGTGCGCGTGGTTCCGGCACGCCGGACGGGTCAGTTGCCGAT CATGGCGAGCAATGCCGGGGTGTACCGCTCCCCGGACACCGGGTGGGAGATCGCGGCCGT CACCTCCGCGAGGGACCGGTCGTCCAGCCGGATCGAGGCGGCGGCGAGATTGTCCGCGAG ATGGGCCGGGTTCGCGGTGCCCGGGATCGGGACGACGTCCTCGCCCCGGTGGTGCAGCCA GGCGAGCGCGAGCTGTGCCAGGGTCAGCCCCAGACCGTCCGCGACCGGGCGCAGCCGGTG CAGCAACGAGCGGTTGCGCGCGAGGGCCGGAGCGCTGAACCGGGGCTGGCCCCGGCGGAA GTCCTCGTCCCCCAGATCGTCGGTGGTGCGGATGGTGCCGGTGAGAAAACCCCGTCCCAG AGGGGCGTAAGCGACGATCCCGATCCCCAGCTCCCGGCAGACGGGCACCACCTCGTCCTC GATCCCGCGCGACCACAGGCTCCACTCGCTCTGCACCGCCGTCACCGGGTGCACCGCGTC CGCCCGGCGCAGCGTGGCCGCGGAGGGCTCGGAGAGACCGAGCCTGCGGACCTTGCCCTC GCGCACCAGCTCGGCCACCGCACCCACGGTCTCCTCGATCGGCACCGCCGGGTCCGTCCA GTGCTGGTAGTACAGGTCGATGCGGTCGGTGCCGAGACGACGCAGGGACCGTTCGCAGGC CGCGCGGACGTAGGACGGCTCGCCGCACAAGCCCTGGGAGGCGCCGTCGGACGAGCGCAC CATGCCGAACTTGGTGGCGATCAGCACCTCGTCCCGGCGGCCCGCGACCGCCCGTCCGAG CAGCTCCTCACCGGCGCCGAGCCCCTGGACGTCGGCGGTGTCCAGCAGGGTGACCCCGGC GTCGACGGCGGCGCGGATGGTGGCCGTCGCCCGGGCGCGGTCCGGGCGTCCGTAGAAGTC GGTGGTCGGCAGGCAGCCGAGCCCCTGGGCACTGACCGGAAGGTCCCGCAGGGCGCGGAC CGGCGGACGCGGAACCGCGGCGGACACGGAACCGGCCGGGGACTCGGGCGGAGAGCGGGA CATACGGAACCTCCACAGGCGGAGCCGGGAACGGGACGAGGGCGAGGACGGGACGGAACG AAGGAGAGGACGGGACGGACAGCACGGACGGGACGGACGGAACGGAGTCGGGAACCGGGG GGGGTGACCGGAACCGGGCCGTCCTTGGCCCTCCCCCGTCCTCCCCGCCATCCGCCGTTC TCCCCCGTTCCCTCTCCCGTCCTCCAGCCAACACCGCCGCCCTTTCCAAGCGCTTGACAC GGCACCGACAGCCGCCGCCGGGCGCCCGATGGGGACCCGTGCCCGCCGGTGAGCGGCGGT GAGCGCCGGTACGGGACCCCACGCGCCGCCGCCCGGGCGCCCGCCAGGGCCCGCGCGGCC ACCCCGGCCCGCCCCGGCCGGAGCGGCGATCCGGGCCGCTCGCTGCAAGAGGAACATCCA CAGCCGCACAAGGAGCGCTCCGCACAGTGGGCACCACGTCCGCCCCGTCCCCCACACCGT GGCCGGTCCCCACCGGACAGCACAGCACCGCACAGCACCACATCGCACGGCACAGCACAG CACCACCGGCACGAGGAACCAAGGAAAGGAACCACACCACCATGACCTCAGTGGACTGCA CCGCGTACGGCCCCGAGCTGCGCGCGCTCGCCGCCCGGCTGCCCCGGACCCCCCGGGCCG ACCTGTACGCCTTCCTGGACGCCGCGCACACAGCCGCCGCCTCGCTCCCCGGCGCCCTCG CCACCGCGCTGGACACCTTCAACGCCGAGGGCAGCGAGGACGGCCATCTGCTGCTGCGCG GCCTCCCGGTGGAGGCCGACGCCGACCTCCCCACCACCCCGAGCAGCACCCCGGCGCCCG AGGACCGCTCCCTGCTGACCATGGAGGCCATGCTCGGACTGGTGGGCCGCCGGCTCGGTC TGCACACGGGGTACCGGGAGCTGCGCTCGGGCACGGTCTACCACGACGTGTACCCGTCGC CCGGCGCGCACCACCTGTCCTCGGAGACCTCCGAGAGGCTGCTGGAGTTCCACACGGAGA TGGCCTACCACCGGCTCCAGCCGAACTACGTCATGCTGGCCTGCTCCCGGGCCGACCACG AGCGCACGGCGGCCACACTCGTCGCCTCGGTCCGCAAGGCGCTGCCCCTGCTGGACGAGA GGACCCGGGCCCGGCTCCTCGACCGGAGGATGCCCTGCTGCGTGGATGTGGCCTTCCGCG GCGGGGTGGACGACCCGGGCGCCATCGCCCAGGTCAAACCGCTCTACGGGGACGCGGACG ATCCCTTCCTCGGGTACGACCGCGAGCTGCTGGCGCCGGAGGACCCCGCGGACAAGGAGG CCGTCGCCGCCCTGTCCAAGGCGCTCGACGAGGTCACGGAGGCGGTGTATCTGGAGCCCG GCGATCTGCTGATCGTCGACAACTTCCGCACCACGCACGCGCGGACGCCGTTCTCGCCCC GCTGGGACGGGAAGGACCGCTGGCTGCACCGCGTCTACATCCGCACCGACCGCAATGGAC AGCTCTCCGGCGGCGAGCGCGCGGGCGACGTCGTCGCCTTCACACCGCGCGGCTGAGCTC CCGGGTCCGACACCGCGCGGCTGAACCCACGGTCCGGGGCCCACGGTCCGGCACCGCGCG GCTGAGCCCCCGGGTCCGGCAGCGGGCGGCTGAACCCCCGCCCCGGGCCACCGCCCGACC GCCCCCGCGCACCGGACGCGCCCGCCTGTACGGCGGTCCCGCCCGGGCCCGTACACCTGA AGCGCCCGGCGGACCGCCGCCCCGCCGGGGGACGGACAGAGCCGGGTGCGGGAGGACGTC CTCCCGCACCCGGCTCCCACCGTTCCGCACCGACCGCACCCGACCGTGCCGCAGGCGCCA CCGGCACCGCACCGCCCGCGCCGGCAGCCACCACAGGCGCCACGCCGCCCGCACGGTGCC CGCGCTGCTCAGCCCCCGTCCACCGGGCTGTCCAGCAGCCGCCGCAGCGCGCCCCCGATG AACTCCCGGTCGGCGGCCGACCCCCCGGACCCCGCGAGATGCCCCCACACTCCCGGGATC ACCTCCAGCGAGGCATACGGCAGCAGATCGGCCACCCGCTTCTCGTCCTCGACGGCGAAA CACACGTCCAGGGCGCCCGGCAGCACCACGGCCCGCGCCGTGACGGAGGCCAGCGCCGCC TCGACGCTCCCCCCGGCCCCGGGTGTCGCCCCCACATCCGTGTTCTCCCAGGTGCGCACC ATGGTGAGCAGATCCGCGGCGCCGGGCCCGGAGAGGAAGACCTGCTCCCAGAAGCCGGTG AGGTACTCCTCGCGGGTGGCGAAACCCAGCTCCCGGTGGGCACGGCGGGCCCAGAAGGAA CGCGAGGTCCCCCACCCGGCGAACACCCGGCCCGCCGCCTTCCGCCCCCGCTCCCCGGCG TCGGCGCTGAGCGCCGCGGCCAGACCGGACAGCAGGACCAGGCTGTGCGGGCTGCTCACC GGCGCCCCGCAGATCGGGGCGATCCGGCGCACCATCCCCGGATGCGACACGGCCCACTGG TAGGCGTGGGCCGCGCCCATCGACCAGCCCGTGACCAGGGCCAGTTCCCGTACCCCCAGC TCCTCGGTGAGCAGCCGGTGCTGCGCCGCGACATTGTCCTGCGGAGTGATCAGCGGAAAG CGGGACCCCGACGGGTGGTTGCCGGGCGAGCTGGAGACCCCGTTGCCGAAGAGTCCGGCG GTGACGACGCAGTACCGCCGGGTGTCCAGCGGCAGCCCCGCACCGATCAGCCAGTCGTAC CCGGTGTGGTCCCGGCCGAAGAACGACGGACAGAGCACCACGTTCGTCCCGTCGGCGTTC GGCGTGCCGTACATGGCGTAACCGATCCGGGCGTCCCGCAGGACCTCCCCGTCCAGCAAC GGCAGTTCGTCGATCTCGAATATGCGGCATTCCACCGCTGACCTCCTTGTTCGATCCCCC CGGACAACAGGTCGGTCGTG GCCGGAGACTCAGAGCCAGTTGGGGGCGATCTCGGTGGCC CACAGCTCCAGGCTGCGCAGCTGGACATCGTGCGGGATCAGCCCGGAGTACTGGCACTGG AGCAGATACTCCGGATCGTGCCGCTCCACCAGCTTCTCGATCATGCGGTTGATGTCGTCC GGGGTGCCGACCCACTCCAGCCCCCGGTCGACCAGGGTCTTGTAGTCCGAGCCGATCGGA CCCGTCTCGCCGGTCGCGCGCAGCGCCTCGGTGAAGCCCATGGGGCCGAACCAGTTCTCG AAGATGAAGCCGCCGCCGCGGGACGCCCAGTGGTGGGCCTCGCCGGAGTCCCGGGAGACC AGGACGTCCTTCATCACCCCGACCCGCTCGCCCCGCCGCAGGGTGCCGTGGCCCGCCGCC TCGGCCTCCTCCCGGTAGATGTCCATCAGCCGGGCGACGATCTGGTCGTCGGTGTTCATC AGGATCGGCACCACGCCCTCCCGGGCACAGAACCGGAACGTGTCCTCACTGAAGCTGAAC GGCTGGAAGACGGGCGGGTGGGGGCGCTGGTAGGGCTTGGGCGCGATGCCCACCTCGCGG ATGACGCCGTTCTCGTCGAGGCCCCGGCCGTAGCGGCGCACCGCCTCGTAGGGGAACTCC AGGTCCGGCACCGGGATCGTCCACTGCTCCCCGGAGTGGGTGAACGTCTCGGTCGTCCAC GCCTTCTTGATGATCTCCCAGTGCTCCTCGAAGAGGGCACGATTGCGCCGGTCCCGCTCC CCGGCGTCGGACAGGGTGCCGCCGACCCCGTACACCTGCCCCATGATGTCGGCCCAGCGC TTCTGGAACCCGCGCGCGATCCCGACGAAGGCGCGGCCCCGGGTCATGTGGTCGAGCATC GCCAGATCCTCGGCCAGCCGCAGCGGATTGTGCAGCGGCAGGACGTTGGCCATCTGGCCG ACCCGGATGTGCCGGGTCTGCATGCCGAGGTAGAGCCCCAGCATGATCGGGTTGTTGGAG ACCTCGAAACCCTCGGTGTGGAAGTGGTGCTCGGTGAAGGACAGTCCCCAGTAGCCGAGT TCGTCGGCCGCCTGCGCCTGCCGGGTGAGCTGCCGGAGCATGTTCTGGTAGTTCTGCGGA TTGACCCCCGCCATACCCCGCTGGACCTGCGCATGACTGCCGACCGTTGGCAGATAGAAG AGAATGGACTTCACCCTGGCTCCTCCGGTTCGCGGCGCCCTCCATTGACGTGCGCCGAAA GCGGCTCGACCGTCCCACTCCGCCCTTGAGTTCCGTCTGACGCCGCGCCAGTCGGCGGGC CGTCCGCCGGGGTGCCCGCCGGGGTCCGCACCCGCCGGACGGCACGGCGCGCACCGCGCG CGCGGCGCTTCGGGGCACCGGGCTCGACGGGGTGCTCAGCGGGACGTCCAACGGAAGGCA AGCCCCCGTACCCAGCCTGGTCAAGGCGCTCATCGCCATTCCCTGAGGAGGTCCCGCCTT GACCACAGCAATCTCCGCGCTCCCGACCGTGCCCGGCTCCGGACTCGAAGCACTGGACCG TGCCACCCTCATCCACCCCACCCTCTCCGGAAACACCGCGGAACGGATCGTGCTGACCTC GGGGTCCGGCAGCCGGGTCCGCGACACCGACGGCCGGGAGTACCTGGACGCGAGCGCCGT CCTCGGGGTGACCCAGGTGGGCCACGGCCGGGCCGAGCTGGCCCGGGTCGCGGCCGAGCA GATGGCCCGGCTGGAGTACTTCCACACCTGGGGGACGATCAGCAACGACCGGGCGGTGGA GCTGGCGGCACGGCTGGTGGGGCTGAGCCCGGAGCCGCTGACCCGCGTCTACTTCACCAG CGGCGGGGCCGAGGGCAACGAGATCGCCCTGCGGATGGCCCGGCTCTACCACCACCGGCG CGGGGAGTCCGCCCGTACCTGGATACTCTCCCGCCGGTCGGCCTACCACGGCGTCGGATA CGGCAGCGGCGGCGTCACCGGCTTCCCCGCCTACCACCAGGGCTTCGGCCCCTCCCTCCC GGACGTCGACTTCCTGACCCCGCCGCAGCCCTACCGCCGGGAGCTGTTCGCCGGTTCCGA CGTCACCGACTTCTGCCTCGCCGAACTGCGCGAGACCATCGACCGGATCGGCCCGGAGCG GATCGCGGCGATGATCGGCGAGCCGATCATGGGCGCGGTCGGCGCCGCGGCCCCGCCCGC CGACTACTGGCCCCGGGTCGCCGAGCTGCTGCACTCCTACGGCATCCTGCTGATCTCCGA CGAGGTGATCACGGGGTACGGGCGCACCGGGCACTGGTTCGCCGCCGACCACTTCGGCGT GGTCCCGGACATCATGGTCACCGCCAAGGGCATCACCTCGGGGTATGTGCCGCACGGCGC CGTCCTGACCACCGAGGCCGTCGCCGACGAGGTCGTCGGCGACCAGGGCTTCCCGGCGGG CTTCACCTACAGCGGCCATGCCACGGCCTGCGCGGTGGCCCTGGCCAACCTGGACATCAT CGAGCGCGAGAATCTGCTCGACAACGCCAGCACCGTCGGCGCCTACCTGGGCAAACGCCT GGCCGAGCTGAGCGATCTGCCGATCGTCGGGGACGTCCGGCAGACCGGTCTGATGCTCGG TGTCGAACTGGTCGCCGACCGCGGAACCCGGGAGCCGCTGCCGGGCGCCGCCGTCGCCGA GGCCCTGCGCGAGCGGGCGGGCATCCTGCTGCGCGCCAACGGCAACGCCCTCATCGTCAA CCCCCCGCTGATCTTCACCCAGGAAGACGCCGACGAACTCGTGGCGGGCCTGCGCTCCGT ACTCGCCCGCACCAGGCCGGACGGCCGGGTGCTCTGACCCCTTTGGCCCTCCCCGGCCCC ACCGGGGCACCACCCCGCCGCACCCCGAGCGCAAAAAGACCCCTCTGCCTGCGTTTCCGC AGGTCAGAGGGGTCTGGTGCAGTGGAGCCTAGGGGAGTCGAACCCCTGACATCTGCCATG CAAAGACAGCGCTCTACCAACTGAGCTAAGGCCCCGAAGCGACAGAACGGCCCTGGACTG CTCCGTCCCGGCCACTGCCGCAGACCAGAGTACCGGGTGTTCCCGGTGATCCTCCAAAAC ATTGAGGTCTCCCGGTGGGCGACCACTCTCCGTAAGATGCTCGACGTGGTTCGCAGCAGC GAAGCCCGCTTGGGGAAGCGATGGGGAGACGCGCATGGACGCCGCTCAGCAGGAGACGAC CGCAAGAGCCCGGGAGCTACAGCGAAGCTGGTACGGGGAGCCCCTGGGGGCCCTGTTCCG CAGGCTGATAGACGATCTGGGGCTGAACCAGGCGCGTCTCGCGGCGGTGCTGGGCCTCTC CGCCCCCATGCTCTCCCAGCTCATGAGCGGCCAGCGGGCCAAGATCGGCAACCCGGCCGT GGTCCAACGGGTCCAGGCGCTCCAGGAGTTGGCCGGACAGGTGGCCGACGGCAGCGTCAG CGCGGTGGAGGCCACCGACCGCATGGAGGAGATCAAGAAGTCGCAGGGAGGCTCCGTCCT GACCGCGAACAGCCAGACCACCAACAGCTCGGGGGCGCCGACCGTCCGCCGGGTCGTCCG GGAGATCCAGTCGCTGCTGCGGTCCGTGTCCGCCGCGGGGGACATCATCGACGCGGCGAA CTCCCTCGCCCCGACCCATCCGGAGCTGGCAGAGTTCCTGCGGGTGTACGGGGCCGGGCG CACCGCGGACGCCGTGGCGCACTACGAGTCCCACCAGAGCTGACGACCGAGGCCGGCCCC GGAACGGACCAGAGCCTCATGAGGGACGGGGAGCGGACGCGGCACCATGGGTGAGGTCTT CGCCGGCCGGTACGAGCTGGTCGACCCGATCGGACGCGGAGGGGTCGGCGCGGTCTGGCG CGCCTGGGACCACCGGCGCCGCCGCTATGTGGCGGCCAAGGTGCTCCAGCAGAGCGACGC GCACACCCTGCTGCGCTTCGTCCGCGAGCAGGCCCTGCGGATCGACCATCCCCATGTCCT GGCCCCGGCGAGCTGGGCCGCGGACGACGACAAAGTCCTCTTCACCATGGATCTCGTGGG CGGCGGATCACTCGCGCACGTGATCGGCGACTACGGCCCGCTCCCGCCGCGCTATGTGTG CGCCCTGCTGGACCAACTCCTCTCCGGGCTCGCCGCGGTGCACGCCGAGGGCGTGGTGCA CCGCGACATCAAACCGGCGAACATCCTGATGGAGGCCACCGGGACGGGCCGCCCCCATCT GCGCCTGTCCGACTTCGGCATCTCCATGCGCAAGGGCGAGCCCCGGCTGACCGAGACCAA CTATGTCGTGGGTACGCCCGGTTACTTCGCCCCCGAGCAGGTCGAGGGCGCGGAGCCGGA CTTCCCCGCCGATCTCTTCGCCGTCGGCCTGGTCGCCCTCTATCTGCTGGAGGGTCAGAA ACCCGACACCAAGGCCCTGGTGGACTTCTTCACCGCCCATGGCACCCCCGGTGCTCCCCG GGGGATACCGGAGCCGCTGTGGCAGGTGCTCGCGGGGCTGATCCAGCCCGACCCCGCCGC CCGGTTCCGTACGGCGACGGGGGCCCGGAAGGCCCTCGCCGCCGCCGTGGAACTGCTTCC CGAGAGCGGCCCCGACGACGAACCGGTGGAGATATTCGACCAACTGGGCCCGCTGCCGCC GGGGTTCGGCCCCGGCGGCCCCGAGAACACGCCGCCCTCCGGTCTGCTGCGCTCGGCGGC CTCCGGTACC
SEQ ID NO: 18 orQpar reverse complement
ATGGCCACCACGACCGCGAAAGCCATGCTGGAACGTCTTCACCAGTACGGTGTCGACCATGTATTCGGCGTCGTCG GCCGGGAGGCGTCCGCCATTCTCTTCGACGAGGTCGAAGGACTCGACTTCGTCCTGACCCGGCACGAGTTCACCGC CGGGGTGATGGCGGACGTCCTCGCCCGGATCACCAACCGCCCCCAGGCGTGCTTCGCGACCCTGGGCCCCGGCATG ACCAACCTGGCCACCGGCGTCGCCACCTCCGCCCTGGACCGCAGCTCGGTCATCGCGCTGGCCGCGCAGTCCGAGT CGTACGACTGCTACCCCAACGTCACCCACCAGTGCCTGGACAGCACCGCCGTGATGGGCCCGCTGACCAAGTTCAG CGTCCAGCTCGAACGCGGCGAGGACATCGTCAACCTCGTCGACAGCGCCGTCCTCAACAGCCGGATCGAGCCCGTG GGTCCCAGCTTCATCAGCCTGCCGGTCGACCTCCTCGGCGCCGAGCTGAACGGCACCCCCACCGACGCCCCCCTGG TCCGGGCCACCGCCACCCACGCCCTGGACGCCGACTGGCGCGCCCGCCTCGACGAGGCCGCTGAGCTGGTGCGCGA GGCCGAGAACCCCCTCCTCGTCGTCGGTAGCGCCGTCATCCGCGCCGGGGCCGTCGACGCCCTGCGCGCCCTCGCC GAGCGGCTGAACATCCCCGTCGTCACCACCTACACCGCCAAGGGCGTCCTGCCGCACGACCACCCGCTCAACTACG GCGCCATCAGCGGCTACATGGACGGCATTCTCGGCCACCCGGCCCTCGACGAGATCTTCGGCCCCGCCGACCTCCT CCTGGCGATCGGCTACGACTACGCCGAGGACCTGCGCCCCTCCATGTGGACGCGGGGCCGGGCCAAGACCACGGTC CGGGTCGCCCCCGAGGTCAACCCGATCCCGGAGCTGTTCCGCGCCGACATCGACATCGTCACCAACGTCGCCGAAT TCGTCACCGCGCTCGACGACGCGACCTCGGGCCTCGCCCCCAAGACCCGGCACGACCTCAGCGCCCTGCGCGCCCG CGTCGCCGAATTCCTCGCCGACCCCACCGAGTACGAGGACGGCATGCGGGTCCACCAGGTGATCGACTGCATGAAC TCCGTCCTCGACAACGGCACCTTCGTCAGCGACATCGGCTTCTTCCGCCACTACGGCGTGCTCTTCGCCAAGTCCG ACCAGCCGTACGGATTCCTCACCTCCGCGGGCTGCTCCAGCTTCGGCTACGGACTGCCCGCCGCCATGGCCGCCCA GATCGCCCGGCCCGGCGAGCCCGTCTTCCTCATCGCGGGCGACGGCGGCTTCCACTCCAACAGCGCCGACATCGAG ACGGCCGTGCGCCTGGGCCTGCCGATCGTCATGGTCGTCGTCAACAACGACCGCAACGGCCTGATCGAGCTGTACC AGAACCTCGGACACCAGCGCTCCCACGCCCCCGCCGTCGGCTTCGGAAGCGTCGACTTCGTCCAGCTCGCCGAGGC CAACGGCTGCGAGGCCGTCCGCGCCACCGACCGCACCTCGCTGCTCGCCGCCCTCACCAAGGGCGCCGGACTCGGC CGCCCGTTCCTGATCGAGGTACCGGTGGCCTACGACTTCCAGTCCGGCGGTTTCGCCGCCCTGGCCATCTGA
SEQ ID NO: 19 orβpar reverse complement
ATGCCCGGCCCCGACCTCGTGTACGGATTCCGGGTGCGCATCGGCACCGAGGGCCGCCCCGGCGGCGGCCCCGGCG GTCACTCCGAACCCGGCAGCGCACCCCGCTTCGCCGTCCGCGGGACCCATGTCCCCGTGCACGACGGCACCGCGTA CCCGCTCTGGAGCGGAACGGCCGTGACCCTGGGCCGTCCGCCCGTCCTGGTCGCCGACGGCCAGGTCCGGCTGCTC CTGGCGGGCGAGCTGTACAACCGCGCCGAGCTGACCGGAGCGCTCGGCGGCTCCTCTGCCGCCCTCGGCGACGCCG AACTGCTGCTGGCCGCCTGGCGGCGCTGGGGCCCCGGGGCCTTCCGGCTCCTGAACGGACGGTTCGCCGCACTGCT CACCGACGCCTCCACCGGCGCGACCGTCGCGGCCACCGACCACGCCGGTTCGGTACCGCTGTGGCTGCGCGCCGAC GTGACGGGGCTGAGCGCCGCCACCGAGGCGAAGACCCTGGCGCACGAGCCGGGCCGGCCGCTGGGCCTGTCCGGCA CCCACACCCGCCGGGGGCGGCGGGCGTCTGCCGGGTCCCCGCCGGGACCGCCCTCCTGCTGCACGGAGTCGGCGGC TCCGACATCACCGCCAGGGCGGTCCGCACCTGGACACCCCCGCTCTCCCGGGCGCTGCCCGGCGAACGGGAGGCGG TGGACCTGGTCGGCGAACGCCTCGCCACGGCGGTCCGCACCCGGCTGCGCGGCGGGGAGGCGGCCCCCACCGTCGT CCTGTCCGGCGGCATCGACTCCGGGGGAGTCGCCGCCCACACGGCGGCCCTGGCACCCGGGACACGGTCCGTGTCG ATGGGCACCGAGGTGTCCGACGAGTTCGACGCGGCCCGCTCGGTCGCCGTCCACCTGGGCACCGCGCACAGCGAGA TCCGGCTCCACTCGGCCGAACTCGTCAGGGAACTGCCCTGGGCGGTCGCCGCCGCGGAGATCACCGACCCCACGGT CCTGGAGTACCTGCTGCCGCTCGTCGCCCTCTACCGGCGGCTCGACACCGGGCCGCTCCGCATCCTCACCGGGTAC GGCGCCGACATCCCGCTCGGCGGTATGCACCGGCGCACGGCCTCGCTCTGGTCCCTCGACGACGAGATCGCGGGCG ACATGGCGGGCTTCGACGGCCTCAACGAGATGTCCCCCGTCCTCGCGGGCATCGCCGGGAAGTGGACCACCCACCC GTACTGGGACCGCGCGGTCCTGGACGCGCTGGTCTCCCTCGAACCCGGGCTCAAACGCCGGCGGGGCACCGACAAG TGGGTGTTGCGGCAGGCCCTCTCCGGCCTGCTGCCCGCCGAGACCGTGGCCCGCCCCAAGCTGGGCATCCACGAGG GGTCCGGCACCACCAGCGCGTGGACCGGACTGCTCCTCGCCGAAGGGATCCGGCGCGACGAGGTGACGGCCGTCAA GGGCGCCATGGCACGGCGCCTGTACGACGCGGTGGTCATCGACACGGTGCCGCCGGAGGACGTGGACTTCGGCGAG ACGGTGCGGCGCTCCGTCGACGCGGTGCGCAGGCTCAGGCTCCAGGGCCGGGTGGTCGTATGA SEQ ID NO:20 orf4par reverse complement
GTGTCCACCGCCGTCTCCCCGCGCTACGCCCAACCGGCGACCTTCATGCGGCTGCGCCACCGGCCCGACCCGATCG GCCATGACGTGGTGGTCGTCGGCGCCCCGTACGACGGAGGCACCAGCTACCGGCCCGGCGCGCGGTTCGCGCCGCG CGCCATCCGGCACGAGTCCAGCCTGATCCACGGCGTCGGCATCGACCGGGGCCCAGGGGTCTTCGACCGGATCGAC GTGGTCGACGGGGGCGACATCGACCTCAGCCCCTTCTCGATGGACCTGGCGATGGACACCGCGACGGTCGCCCTGA CCCGGCTCCTGGAACGCAACGACGCGTTCCTGATGCTGGGCGGGGACCACTCGCTCTCCCTGGCCGCCCTGCGCGC CGTGCACGCCCGCCACGGCCGGGTCGCCGTCCTGCACCTGGACGCGCACAGCGACACCAACCCACCCGTCTACGGC GGCACCTACCACCACGGCACCCCCTTCCGCTGGGCCATCGAAGAGGGCCTGGTGGACCCGGAGCGCCTGGTCCAGG TCGGCATCCGCGGCCACAATCCGCGGCCCGACTCCCTGGACTACGCGCGCGGGCACGGCGTCAGCATCGTCACCGC CGCCGACTTCACCCGGCGCTCACCGCGCGGCATCGCCGAGCAGATCCGGCGCACCGTCGGCGGCCTGCCGCTGTAC GTCTCCGTCGACATCGACGTCGTCGACCCGGCGTACGCCCCGGGCACCGGCACACCGGCCCCCGGCGGGCTGTCCT CGCGCGAGGTGCTGACCCTGCTCGACGTGGTCGGGCAGCTCAGGCCCGTCGGCTTCGACGTGGTCGAGGTGTCCCC GGCGTACGACCCGTCGGGGATCACCTCCCTGCTGGCGGCGGAGATCGGGGCCGAACTGCTCTACCAGTACGCCCGC GCCACCACGTCGCCCGCGTCGGCACCGGTGGACTCTCCCCTGCCACCGGGGGCGGCGGCGGACGACGCCGAGAACG CCGAGAACGCGGTGGACGCGGTGGACGCCGAGAGCGCCGTGGACTTCGCCGGGCAGCGGTGGGGGTAG
SEQ ID NO:21 cvmό Polypeptide VPGSG EALDRATLIHPTLSGNTAERIVLTSGSGSRVRDTDGREYLDASAV GVTQVGHGRAELARVAAEQMARLEY FHTWGTISNDRAVELAARLVG SPEP TRVYFTSGGAEGNEIALRMARLYHHRRGESART I SRRSAYHGVGYGSG GVTGFPAYHQGFGPSLPDVDFLTPPQPYRRELFAGSDVTDFCLAE RETIDRIGPERIAAMIGEPIMGAVGAAAPPA DYWPRVAEL HSYGILLISDEVITGYGRTGHWFAADHFGVVPDI VTAKGITSGYVPHGAVLTTEAVADEVVGDQGF PAGFTYSGHATACAVALANLDIIERENLLDNASTVGAYLGKR AELSD PIVGDVRQTGLMLGVELVARGTREPLPG AAVAEALRERAGILLRANGNALIVNPPLIFTQEDADELVAGLRSVLARTRPDGRV
SEQ ID NO:22 cvm3 Polypeptide
VTRPPG SAHTHGΞVSGS LRRVAGHYPTGWLVTGPAEAPGQPPPAMWGTFTSVSLDPVLVGFLPARSSTTWPR LRAAGRFCVNVLGADQGPVCRSFAGGDPGRWEVPYRTTATGSPV DA A FDCEVAGETEAGDHWFVTGAVRDLG VIREGS PLVFLRGDYGH AGGGGSGRAGRRS AVCPV
SEQ ID NO:23 orfόpar Polypeptide RASSPRGFRVHHGHAGIRGSHADLAVIASDVPAAVGAVFTRSRFAAPSVLLSRDAVADGIARGVVV SGNANAGT GPRGYEDAAEVRH VAGIVDCDERDVLIASTGPVGERYPMSRVRAHLRAVRGPLPGADFDGAAAAVLGTAGARPTI RRARCGDATLIGVAKGPGTGPAEQDDRSTLAFFCTDAQVSPVVLDDIFRRVADRAFHGLGFGADASTGDTAAVLAN GLAGRVDLVAFEQVLGALALD VRDVVRDSGCGGALVTVRVTGAHDTEQAGRVGRAVVDAPSLRAAVHGPAPDWAP VAAVAGGHGDEGPGRSPGRITIRVGGREVFPAPRDRARPDAVTAYPHGGEVTVHIDLGVPGRAPGAFTVHGCDLLA GYPRLGAGRAV. SEQ ID NO:24 orf4par Polypeptide
VSTAVSPRYAQPATFMRLRHRPDPIGHDVVWGAPYDGGTSYRPGARFAPRAIRHESSLIHGVGIDRGPGVFDRID VVDGGDIDLSPFSMDLAMDTATVALTRLLERNDAFL LGGDHSLSLAALRAVHARHGRVAVLHLDAHSDTNPPVYG GTYHHGTPFRWAIEEGLVDPERLVQVGIRGHNPRPDSLDYARGHGVSIVTAADFTRRSPRGIAEQIRRTVGGLPLY VSVDIDVVDPAYAPGTGTPAPGGLSSREVLT LDVVGQLRPVGFDVVEVSPAYDPSGITSLLAAEIGAELLYQYAR ATTSPASAPVDSPLPPGAAADDAENAENAVDAVDAESAVDFAGQRWG.
SEQ ID NO:25 orβpar Polypeptide
MPGPDLVYGFRVRIGTEGRPGGGPGGHSEPGSAPRFAVRGTHVPVHDGTAYPL SGTAVT GRPPV VADGQVRLL LAGELYNRAELTGALGGSSAALGDAELLLAA RRWGPGAFRLLNGRFAA TDASTGATVAATDHAGSVP WLRAD VTGLSAATEAKTLAHEPGRPLGLSGTHTAPGAAGVCRVPAGTALLLHGVGGSDITARAVRT TPPLSRALPGEREA VDLVGERLATAVRTRLRGGEAAPTVVLSGGIDSGGVAAHTAALAPGTRSVS GTEVSDEFDAARSVAVHLGTAHSE IRLHSAELVRELPWAVAAAEITDPTVLEYLLPLVA YRRLDTGPLRILTGYGADIPLGGMHRRTAS SLDDEIAG DMAGFDGLNEMSPVLAGIAGKWTTHPY DRAVLDALVS EPGLKRRRGTDK VLRQALSG PAETVARPKLGIHE GSGTTSA TGLLLAEGIRRDEVTAVKGAMARRLYDAVVIDTVPPEDVDFGETVRRSVDAVRR RLQGRVVV.
SEQ ID NO:26 orf2par Polypeptide
MATTTAKAM ERLHQYGVDHVFGVVGREASAILFDEVEGLDFVLTRHEFTAGVMADVLARITNRPQACFATLGPG TNLATGVATSALDRSSVIA AAQSESYDCYPNVTHQCLDSTAVMGPLTKFSVQLERGEDIVNLVDSAVLNSRIEPV GPSFISLPVDLLGAELNGTPTDAPLVRATATHA DADWRARLDEAAELVREAENPLLVVGSAVIRAGAVDALRALA ERLNIPVVTTYTAKGVLPHDHP NYGAISGY DGILGHPALDEIFGPADLLLAIGYDYAEDLRPS TRGRAKTTV RVAPEVNPIPELFRADIDIVTNVAEFVTALDDATSG APKTRHDLSALRARVAEF ADPTEYEDGMRVHQVIDCMN SVLDNGTFVSDIGFFRHYGVLFAKSDQPYGFLTSAGCSSFGYGLPAAMAAQIARPGEPVFLIAGDGGFHSNSADIE TAVRLG PIVMVVVNNDRNGLIELYQNLGHQRSHAPAVGFGSVDFVQLAEANGCEAVRATDRTS LAALTKGAG G RPFLIEVPVAYDFQSGGFAALAI

Claims

Claims
1. A S. clavuligerus microorganism comprising DNA corresponding to one or more open reading frames essential for 5S clavam biosynthesis, wherein said open reading frames are disrupted or deleted such that the production of 5S clavams by said S. clavuligerus is reduced and clavulanic acid production is at least maintained, wherein the open reading frames are selected from: a) cvmόpara (SEQ ID NO: 1); b) cvmlpara (SEQ ID NO:2); c) cvmόpara and cvmό (SEQ ID NO:5); or d) cvmlpara and cvml (SEQ ID NO:6).
2 A S. clavuligerus microorganism comprising DNA corresponding to one or more open reading frames essential for 5S clavam biosynthesis, wherein said open reading frames are disrupted or deleted such that the production of 5S clavams by said S. clavuligerus is reduced and clavulanic acid production is at least maintained, wherein the open reading frames are selected from: a) cvmόpara and one or more of cvml (SEQ ID NO:7), cvm2 (SEQ ID NO: 8), cvm3 (SEQ ID NO:9), cvm4 (SEQ ID NO: 10), cvm5 (SEQ ID NO: 11), cvmό, cvml or cvmlpara; or b) cvmlpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvmό, cvml or cvmόpara.
3. An isolated polynucleotide comprising open reading frames selected from the group consisting of: a) cvrnόpara; b) cvmlpara; c) cvmόpara and cvmό; d) cvmlpara and cvml; e) cvmόpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvmό, cvml or cvmlpara; or f) cvmlpara and one or more of cvml, cvm2, cvm3, cvm4, cvm5, cvmό, cvml or cvmόpara.
4. An isolated polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames are selected from the group consisting of: a) orβpara (SEQ ID NO: 12), b) orβpara (SEQ ID NO: 13), c) orβpara (SEQ ID NO: 14), and d) orfόpara (SEQ ID NO: 15).
5. An isolated polynucleotide comprising one or more open reading frames encoding one or more enzymes involved in clavulanic acid biosynthesis wherein said open reading frames comprise one or more of: a) orβpara, b) orβpara, c) orβpara, d) orfόpara in combination with one or more genes involved in clavulanic acid biosynthesis selected from orβ, orβ, orβ, orβ, orfό, orβ, orβ, orβ, orf 10, orf 11, orf 12, orf 13, orf 14, orf 15, orflό, orf 11, or orf 18.
6. An isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 16; b) a polynucleotide having the nucleotide sequence of SEQ ID NO: 16; c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17; and d) a polynucleotide having the nucleotide sequence of SEQ ID NO: 17.
7. A vector comprising the polynucleotide of any one of claims 3 to 6.
8. A S. clavuligerus microorganism comprising the vector of claim 7.
9. A process for improving clavulanic acid production in a suitable microorganism comprising isolating the polynucleotide of any one of claims 3 to 6, manipulating said polynucleotide, introducing the manipulated polynucleotide into a said suitable microorgansim and fermenting said suitable microorganism under conditions whereby clavulanic acid is produced.
10. A process according to claim 9 wherein the polynucleotide is a cvm or cvmpara polynucleotide and the manipulation comprises disrupting or deleting cvm or cvmpara gene sequences.
11. A process according to claim 9 wherein the polynucleotide is an orf or orfpara polynucleotides and manipulation thereof comprises insertion of the polynucleotide into vectors suitable for expression.
12. A process according to any one of claims 9 to 11 wherein the suitable microorganism is S. clavuligerus
EP04727009A 2003-04-15 2004-04-13 New process for improving the manufacture of clavams e.g. clavulanic acid Withdrawn EP1613780A2 (en)

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WO2012043622A1 (en) * 2010-09-28 2012-04-05 株式会社カネカ Novel aminotransferase and gene encoding same, and use of the aminotransferase and the gene
EP2589663A1 (en) 2011-11-04 2013-05-08 LEK Pharmaceuticals d.d. Process for production of clavulanic acid

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JPS589679B2 (en) 1977-02-24 1983-02-22 武田薬品工業株式会社 Antibiotic manufacturing method
US4202819A (en) 1979-05-04 1980-05-13 Hoffman-La Roche Inc. 2-(3-Alanyl)clavam antibiotic
GB9020683D0 (en) 1990-09-22 1990-11-07 Univ Strathclyde Isolation of dna
CA2108113C (en) 1993-10-08 2006-12-05 Susan E. Jensen Dna sequence encoding enzymes of clavulanic acid biosynthesis
GB9702218D0 (en) * 1997-02-04 1997-03-26 Smithkline Beecham Plc Novel product
GB9815666D0 (en) * 1998-07-17 1998-09-16 Smithkline Beecham Plc Novel compounds
WO2001030977A2 (en) 1999-10-22 2001-05-03 Smithkline Beecham P.L.C. 5r clavam producing microorganisms with deleted lat and cvm genes
JP2005508189A (en) 2001-11-07 2005-03-31 スミスクライン　ビーチャム　パブリック　リミテッド　カンパニー New molecule

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